Therapeutic agents and uses thereof

ABSTRACT

Human therapeutic compositions are provided, comprising compounds including a plurality of fused polycyclic moieties and a linker moiety. In certain embodiments, the compounds are the reaction products of aldehyde and harmaline components. The compositions exhibit anti-cancer properties, especially against lymphoma, leukemia, pancreatic, endometrial, ovarian, gastric, breast, renal, cervical, head and neck, and myeloma cell lines.

CROSS-REFERENCE TO RELATED APPLICATION

This is a continuation of U.S. patent application Ser. No. 16/935,079 filed Jul. 21, 2020, which is incorporated by reference herein in its entirety.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to chemotherapeutics for treatment of humans, and especially for the treatment of human cancers, and corresponding methods for the treatment of humans suffering from cancers or other maladies. The invention further provides dosage forms and regimens for administration to human patients, and methods of formulating and administering such dosage forms to yield improvements in treatment outcomes. More particularly, the invention is concerned with the administration of specific chemotherapeutic dosage forms (e.g., liquid mixtures, capsules, pills, or tablets) comprising compounds or agents having a plurality of fused polycyclic moieties linked or tethered by means of an appropriate linker. In certain embodiments, compounds having plural β-carboline component moieties and a single linker moiety are provided.

Description of Related Art

Cancer is a generic term for a large group of diseases that can affect any part of the body. Other terms used are malignant tumors and neoplasms. One defining feature of cancer is the rapid creation of abnormal cells that grow beyond their usual boundaries, and which can then invade adjoining parts of the body and spread to other organs. This process is referred to as metastasis. Metastases are the major cause of death from cancer.

The transformation from a normal cell into a tumor cell is a multistage process, typically a progression from a pre-cancerous lesion to malignant tumors. These changes are the result of the interaction between a person's genetic factors and three categories of external agents, including:

-   -   physical carcinogens, such as ultraviolet and ionizing radiation     -   chemical carcinogens, such as asbestos, components of tobacco         smoke, aflatoxin (a food contaminant) and arsenic (a drinking         water contaminant)     -   biological carcinogens, such as infections from certain viruses,         bacteria, or parasites.

Some examples of infections associated with certain cancers:

-   -   Viruses: hepatitis B and liver cancer, Human Papilloma Virus         (HPV) and cervical cancer, and human immunodeficiency virus         (HIV) and Kaposi sarcoma.     -   Bacteria: Helicobacter pylori and stomach cancer.     -   Parasites: schistosomiasis and bladder cancer.

Aging is another fundamental factor for the development of cancer. The incidence of cancer rises dramatically with age, most likely due to a buildup of risks for specific cancers that increase with age. The overall risk accumulation is combined with the tendency for cellular repair mechanisms to be less effective as a person grows older.

Tobacco use, alcohol use, low fruit and vegetable intake, and chronic infections from hepatitis B (HBV), hepatitis C virus (HCV) and some types of Human Papilloma Virus (HPV) are leading risk factors for cancer in low- and middle-income countries. Cervical cancer, which is caused by HPV, is a leading cause of cancer death among women in low-income countries. In high-income countries, tobacco use, alcohol use, and being overweight or obese are major risk factors for cancer.

The most common cancer treatment modalities are surgery, chemotherapy, and radiation treatments. All of these techniques have significant drawbacks in terms of side effects and patient discomfort. For example, chemotherapy may result in significant decreases in white blood cell count (neutropenia), red blood cell count (anemia), and platelet count (thrombocytopenia). This can result in pain, diarrhea, constipation, mouth sores, hair loss, nausea, and vomiting.

Biological therapy (sometimes called immunotherapy, biotherapy, or biological response modifier therapy) is a relatively new addition to the family of cancer treatments. Biological therapies use the body's immune system, either directly or indirectly, to fight cancer or to lessen the side effects that may be caused by some cancer treatments.

During chemotherapies involving multiple-drug treatments, adverse drug events are common, and indeed toxicities related to drug-drug interactions are one of the leading causes of hospitalizations in the US. Obach, R. S. “Drug-Drug Interactions: An Important Negative Attribute in Drugs.” Drugs Today 39.5 (2003): 308-338. In fact, in any single-month period, one-fifth of all surveyed adults in the USA reported an adverse drug response. Hakkarainen, K. M. et al. “Prevalence and Perceived Preventability of Self-Reported Adverse Drug Events—A Population-Based Survey of 7,099 Adults.” PLoS One 8.9 (2013): e73166. A large-scale study of adults aged 57-85 found that 29% were taking more than five prescription medications and nearly 5% were at risk of major adverse drug-drug interactions. In the field of oncology, a review of over 400 cancer patients determined that 77% were taking drugs that were considered to have a moderately severe potential for adverse drug interactions, and 9% had major adverse drug interactions. Ghalib, M. S. et al. “Alterations of Chemotherapeutic Pharmocokinetic Profiles by Drug-Drug Interactions.” Expert Opin. Drug Metabl. Toxicol 5.2 (2009): 109-130.

Such interactions are a global health problem, and the WHO has determined that negative drug interactions are leading causes of morbidity and mortality around the world, with up to 7% of all hospitalizations in the US due to negative drug interactions. A recent survey of a single hospital shows that 83% of hospitalized patients were prescribed drug combinations with the potential to cause adverse reactions. Patel, P. S. et al. “A Study of Potential Adverse Drug-Drug Interactions Among Prescribed Drugs in a Medicine Outpatient Department of a Tertiary Care Teaching Hospital.” J. Basic Clin. Pharm. 5.2 (2014): 44-48.

Examples of famous negative drug interactions include the development of rhabdomyolysis, a severe muscle disease, when taking Simvastatin with Amiodarone. As a result, the FDA introduced a warning on the drug label about the interaction. The calcium channel blocker Mibefradif, taken for high blood pressure, was removed from the market because of the harmful interaction with drugs that work on the electrical activity of the heart.

U.S. Pat. No. 8,039,025 describes cancer treatments in the form of extracts of Arum palaestinum Boiss, supplemented with individual amounts of β-sitosterol, isovanillin, and linoleic acid, and this patent is incorporated by reference herein in its entirety.

U.S. Pat. No. 9,402,834, issued Aug. 2, 2016, describes anti-cancer compositions containing various components in mixtures, such as curcumin, harmine, and isovanillin component mixtures, or component mixtures containing curcumin/harmine, curcumin/isovanillin, and harmine/isovanillin components.

Despite the immense amount of worldwide research and efforts to stem the tide of cancer and its side effects, the disease in its many manifestations continues to be a huge problem. Therefore, any new cancer treatment having a curative affect and/or the ability to ameliorate cancer symptoms and improve the lifestyle of patients is highly significant and important.

SUMMARY OF THE INVENTION

The present invention provides compositions which may be used as improved chemotherapeutics for treatment of humans, and especially in the treatment of human cancers, and corresponding methods for preparing such compositions and use thereof. Generally speaking, the chemotherapeutics of the invention comprise (or consist essentially of, or consist of) one or more compounds and related versions thereof. Thus, as used herein in the present specification and claims, a defined “therapeutic compound” or “compound” means the defined compound per se, as well as the dimers, isomers, tautomers, derivatives, solvates, metabolites, esters, metal complexes (e.g., Cu, Fe, Zn, Pt, V), prodrugs, and salts thereof. Hence, “dimers” refers to a molecule or molecular complex made up of two identical molecules linked together by bonds that can be strong or weak (e.g., covalent or hydrogen bonds); “isomers” refers to each of two or more compounds with the same formula but with at different arrangement of atoms, and includes structural isomers and stereoisomers (e.g., geometric isomers and enantiomers); “tautomers” refers to two or more isometric compounds that exist in equilibrium, such as keto-enol and imine and enamine tautomers; “derivatives” refers to compounds that can be imagined to arise or actually be synthesized from a defined parent compound by replacement of one atom with another atom or a group of atoms; “solvates” refers to interaction with a defined compound with a solvent to form a stabilized solute species; “metabolites” refers to a defined compound which has been metabolized in vivo by digestion or other bodily chemical processes; and “prodrugs” refers to defined compound which has been generated by a metabolic process. The compounds can be directly used in partial or essentially completely purified forms, or can be modified as indicated above. The compounds may be in crystalline or amorphous forms, and may be lyophilized.

The invention also provides new methods for treatment of cancers by administration of appropriate quantities of compositions comprising therapeutic compounds as described herein. Hence, the compositions are particularly designed for use in the treatment of cancers, and the compositions can be used for the manufacture of medicaments for anti-cancer therapeutic applications. In addition, the invention provides compositions for the treatment of cancers comprising administering therapeutically effective amounts of the new compositions, prepared by processes known per se, with a pharmaceutically acceptable carrier.

A “chemotherapeutic,” “chemotherapeutic agent,” or simply “therapeutic agent,” as used herein refers to one or more of the compounds described herein as useful in the treatment of human conditions, especially human cancers. Chemotherapeutics may be cytostatic, selectively toxic, or destructive of cancerous tissue and/or cells, including cancer stem cells, but also include indiscriminately cytotoxic compounds used in cancer treatments.

The therapeutic compounds or agents of the invention have been found to be effective in the treatment of a number of human cancer cells, and especially lymphoma, leukemia, pancreatic, endometrial, ovarian, gastric, breast, renal, cervical, head and neck, and myeloma.

The compounds or agents of the invention broadly comprise a plurality of fused polycyclic moieties linked or tethered by an appropriate linker; preferably, there are two tricyclic moieties. The polycyclic moieties each include at least one N-containing ring. β-carboline moieties are particularly useful in the invention, such as harmaline or similar moieties. In certain embodiments, a pair of β-carboline moieties are bonded by means of a linker moiety, and specifically through a single atom forming at least a part of the overall linker. The reaction products of β-carboline compounds and aldehyde compounds yield a number of useful anti-cancer compounds in accordance with the invention. While the compounds per se of the invention not a part of anti-cancer compositions do not include compounds made up of the reaction product of two harmaline moieties or two harmine moieties, with a linker moiety of benzaldehyde of p-nitro benzaldehyde, the anti-cancer compositions of the invention (which normally include at least one other agent, component, or compound) and treatment methods do embrace such compounds.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph of cell number versus dosage amounts of an orthovanillin/harmaline compound (GZ523.001), illustrating the effect thereof in inducing the death of lymphoma cells, as described in Example 2:

FIG. 2 is a graph of cell number versus dosage amounts of an orthovanillin/harmaline compound (GZ523.002), illustrating the effect thereof in inducing the death of lymphoma cells, as described in Example 2:

FIG. 3 is a graph of cell number versus dosage amounts of an orthovanillin/harmaline compound (GZ523.003), illustrating the effect thereof in inducing the death of lymphoma cells, as described in Example 2:

FIG. 4 is a graph of cell number versus dosage amounts of an orthovanillin/harmaline compound (GZ523.004), illustrating the effect thereof in inducing the death of lymphoma cells, as described in Example 2:

FIG. 5 is a graph of cell number versus dosage amounts of an orthovanillin/harmaline compound (GZ523.005), illustrating the effect thereof in inducing the death of lymphoma cells, as described in Example 2:

FIG. 6 is a graph of cell number versus dosage amounts of an orthovanillin/harmaline compound (GZ523.006), illustrating the effect thereof in inducing the death of lymphoma cells, as described in Example 2:

FIG. 7 is a graph of cell number versus dosage amounts of an orthovanillin/harmaline compound (GZ523.007), illustrating the effect thereof in inducing the death of lymphoma cells, as described in Example 2:

FIG. 8 is a graph of cell number versus dosage amounts of an orthovanillin/harmaline compound (GZ523.008), illustrating the effect thereof in inducing the death of lymphoma cells, as described in Example 2:

FIG. 9 is a graph of cell number versus dosage amounts of an orthovanillin/harmaline compound (GZ523.001), illustrating the effect thereof in inducing the death of leukemia cells, as described in Example 2:

FIG. 10 is a graph of cell number versus dosage amounts of an orthovanillin/harmaline compound (GZ523.002), illustrating the effect thereof in inducing the death of leukemia cells, as described in Example 2:

FIG. 11 is a graph of cell number versus dosage amounts of an orthovanillin/harmaline compound (GZ523.003), illustrating the effect thereof in inducing the death of leukemia cells, as described in Example 2:

FIG. 12 is a graph of cell number versus dosage amounts of an orthovanillin/harmaline compound (GZ523.004), illustrating the effect thereof in inducing the death of leukemia cells, as described in Example 2:

FIG. 13 is a graph of cell number versus dosage amounts of an orthovanillin/harmaline compound (GZ523.005), illustrating the effect thereof in inducing the death of leukemia cells, as described in Example 2:

FIG. 14 is a graph of cell number versus dosage amounts of an orthovanillin/harmaline compound (GZ523.006), illustrating the effect thereof in inducing the death of leukemia cells, as described in Example 2:

FIG. 15 is a graph of cell number versus dosage amounts of an orthovanillin/harmaline compound (GZ523.007), illustrating the effect thereof in inducing the death of leukemia cells, as described in Example 2:

FIG. 16 is a graph of cell number versus dosage amounts of an orthovanillin/harmaline compound (GZ523.008), illustrating the effect thereof in inducing the death of leukemia cells, as described in Example 2:

FIG. 17 is a graph of cell number versus dosage amounts of a composition containing high molecular weight dioligomer compound(s) derived from an orthovanillin/harmaline reaction, illustrating the effect thereof in inducing the death of lymphoma cells, as described in Example 3;

FIG. 18 is a graph of cell number versus dosage amounts of a composition containing high molecular weight dioligomer compound(s) derived from an orthovanillin/harmaline reaction, illustrating the effect thereof in inducing the death of leukemia cells, as described in Example 3;

FIG. 19 is a graph of cell number versus dosage amounts of a vanillin/harmaline compound (GZ518.000), illustrating the effect thereof in inducing the death of lymphoma cells, as described in Example 5;

FIG. 20 is a graph of cell number versus dosage amounts of a vanillin/harmaline compound (GZ518.001), illustrating the effect thereof in inducing the death of lymphoma cells, as described in Example 5;

FIG. 21 is a bar graph illustrating the EC₅₀ values determined by treatment of a plurality of lymphoma cell lines with GZ523.006, as explained in Example 6;

FIG. 22 is a bar graph depicting the results of comparative Caspase 3/7 assays using GZ523.006 against a number of lymphoma cell lines, confirming the cytotoxic properties of GZ523.006 through induction of apoptosis;

FIG. 23 is a graph illustrating cell growth as a function of the concentration of the 518B562 compound, in the MIA PaCa-2 cell proliferation assay described in Example 8;

FIG. 24 is a graph illustrating cell growth as a function of the concentration of the 518B562 compound, in the ASPC-1 cell proliferation assay described in Example 8;

FIG. 25 is a graph illustrating cell growth as a function of the concentration of the 518B562 compound, in the BxPC-3 cell proliferation assay described in Example 8;

FIG. 26 is a graph illustrating cell growth as a function of the concentration of the 518B562 compound, in the AN3CA cell proliferation assay described in Example 8;

FIG. 27 is a graph illustrating cell growth as a function of the concentration of the 518B562 compound, in the HEC-1a cell proliferation assay described in Example 8;

FIG. 28 is a graph illustrating cell growth as a function of the concentration of the 518B562 compound, in the MDA-MB-231 cell proliferation assay described in Example 8;

FIG. 29 is a graph illustrating cell growth as a function of the concentration of the 518B562 compound, in the MDA-MB-468 cell proliferation assay described in Example 8;

FIG. 30 is a graph illustrating cell growth as a function of the concentration of the 518B562 compound, in the HCC70 cell proliferation assay described in Example 8;

FIG. 31 is a graph illustrating cell growth as a function of the concentration of the 518B562 compound, in the H1975 cell proliferation assay described in Example 8;

FIG. 32 is a graph illustrating cell growth as a function of the concentration of the 518B562 compound, in the H1650 cell proliferation assay described in Example 8;

FIG. 33 is a graph illustrating cell growth as a function of the concentration of the 518B562 compound, in the A2780 cell proliferation assay described in Example 8;

FIG. 34 is a graph illustrating cell growth as a function of the concentration of the 518B562 compound, in the A2780CP cell proliferation assay described in Example 8;

FIG. 35 is a graph illustrating cell growth as a function of the concentration of the 518B562 compound, in the RXF-393 cell proliferation assay described in Example 8;

FIG. 36 is a graph illustrating cell growth as a function of the concentration of the 518B562 compound, in the A498 cell proliferation assay described in Example 8;

FIG. 37 is a graph illustrating cell growth as a function of the concentration of the 518B562 compound, in the N87 cell proliferation assay described in Example 8;

FIG. 38 is a graph illustrating cell growth as a function of the concentration of the 518B562 compound, in the SiHA cell proliferation assay described in Example 8;

FIG. 39 is a graph illustrating cell growth as a function of the concentration of the 518B562 compound, in the FaDu cell proliferation assay described in Example 8;

FIG. 40 is a graph illustrating cell growth as a function of the concentration of the 518B562 compound, in the DOHH-2 cell proliferation assay described in Example 8;

FIG. 41 is a graph illustrating cell growth as a function of the concentration of the 518B562 compound, in the SU-DHL-4 cell proliferation assay described in Example 8;

FIG. 42 is a graph illustrating cell growth as a function of the concentration of the 518B562 compound, in the OCI-LY3 cell proliferation assay described in Example 8;

FIG. 43 is a graph illustrating cell growth as a function of the concentration of the 518B562 compound, in the JIM1 cell proliferation assay described in Example 8;

FIG. 44 is a graph illustrating cell growth as a function of the concentration of the 518B562 compound, in the KMM-1 cell proliferation assay described in Example 8;

FIG. 45 is a graph illustrating cell growth as a function of the concentration of the 518B562 compound, in the KMS-11 cell proliferation assay described in Example 8;

FIG. 46 is a graph illustrating cell growth as a function of the concentration of the 518B562 compound, in the KMS-27 cell proliferation assay described in Example 8;

FIG. 47 is a graph illustrating cell growth as a function of the concentration of the 518B562 compound, in the KMS-34 cell proliferation assay described in Example 8;

FIG. 48 is a graph illustrating cell growth as a function of the concentration of the 518B562 compound, in the H929 cell proliferation assay described in Example 8;

FIG. 49 is a graph illustrating cell growth as a function of the concentration of the 518B562 compound, in the L363 cell proliferation assay described in Example 8;

FIG. 50 is a graph illustrating cell growth as a function of the concentration of the 518B562 compound, in the MM.1s cell proliferation assay described in Example 8;

FIG. 51 is a graph illustrating cell growth as a function of the concentration of the 518B562 compound, in the MOLP-8 cell proliferation assay described in Example 8;

FIG. 52 is a graph illustrating cell growth as a function of the concentration of the 518B562 compound, in the Jeko-1 Parental cell proliferation assay described in Example 8;

FIG. 53 is a graph illustrating cell growth as a function of the concentration of the 518B562 compound, in the Jeko-1 Lenalidomine Resistant cell proliferation assay described in Example 8;

FIG. 54 is a graph illustrating cell growth as a function of the concentration of the 518B562 compound, in the Jeko-1 Bortezomib Resistant cell proliferation assay described in Example 8;

FIG. 55 is a graph illustrating cell growth as a function of the concentration of the 560 compound, in the MIA PaCa-2 cell proliferation assay described in Example 8;

FIG. 56 is a graph illustrating cell growth as a function of the concentration of the 560 compound, in the ASPC-1 cell proliferation assay described in Example 8;

FIG. 57 is a graph illustrating cell growth as a function of the concentration of the 560 compound, in the BxPC-3 cell proliferation assay described in Example 8;

FIG. 58 is a graph illustrating cell growth as a function of the concentration of the 560 compound, in the AN3CA cell proliferation assay described in Example 8;

FIG. 59 is a graph illustrating cell growth as a function of the concentration of the 560 compound, in the HEC-1a cell proliferation assay described in Example 8;

FIG. 60 is a graph illustrating cell growth as a function of the concentration of the 560 compound, in the MDA-MB-231 cell proliferation assay described in Example 8;

FIG. 61 is a graph illustrating cell growth as a function of the concentration of the 560 compound, in the MDA-MB-468 cell proliferation assay described in Example 8;

FIG. 62 is a graph illustrating cell growth as a function of the concentration of the 560 compound, in the HCC70 cell proliferation assay described in Example 8;

FIG. 63 is a graph illustrating cell growth as a function of the concentration of the 560 compound, in the H1975 cell proliferation assay described in Example 8;

FIG. 64 is a graph illustrating cell growth as a function of the concentration of the 560 compound, in the H1650 cell proliferation assay described in Example 8;

FIG. 65 is a graph illustrating cell growth as a function of the concentration of the 560 compound, in the A2780 cell proliferation assay described in Example 8;

FIG. 66 is a graph illustrating cell growth as a function of the concentration of the 560 compound, in the A2780CP cell proliferation assay described in Example 8;

FIG. 67 is a graph illustrating cell growth as a function of the concentration of the 560 compound, in the RXF-393 cell proliferation assay described in Example 8;

FIG. 68 is a graph illustrating cell growth as a function of the concentration of the 560 compound, in the A498 cell proliferation assay described in Example 8;

FIG. 69 is a graph illustrating cell growth as a function of the concentration of the 560 compound, in the N87 cell proliferation assay described in Example 8;

FIG. 70 is a graph illustrating cell growth as a function of the concentration of the 560 compound, in the SiHA cell proliferation assay described in Example 8;

FIG. 71 is a graph illustrating cell growth as a function of the concentration of the 560 compound, in the FaDu cell proliferation assay described in Example 8;

FIG. 72 is a graph illustrating cell growth as a function of the concentration of the 560 compound, in the DOHH-2 cell proliferation assay described in Example 8;

FIG. 73 is a graph illustrating cell growth as a function of the concentration of the 560 compound, in the SU-DHL-4 cell proliferation assay described in Example 8;

FIG. 74 is a graph illustrating cell growth as a function of the concentration of the 560 compound, in the OCI-LY3 cell proliferation assay described in Example 8;

FIG. 75 is a graph illustrating cell growth as a function of the concentration of the 560 compound, in the JIM1 cell proliferation assay described in Example 8;

FIG. 76 is a graph illustrating cell growth as a function of the concentration of the 560 compound, in the KMM-1 cell proliferation assay described in Example 8;

FIG. 77 is a graph illustrating cell growth as a function of the concentration of the 560 compound, in the KMS-11 cell proliferation assay described in Example 8;

FIG. 78 is a graph illustrating cell growth as a function of the concentration of the 560 compound, in the KMS-27 cell proliferation assay described in Example 8;

FIG. 79 is a graph illustrating cell growth as a function of the concentration of the 560 compound, in the KMS-34 cell proliferation assay described in Example 8;

FIG. 80 is a graph illustrating cell growth as a function of the concentration of the 560 compound, in the H929 cell proliferation assay described in Example 8;

FIG. 81 is a graph illustrating cell growth as a function of the concentration of the 560 compound, in the L363 cell proliferation assay described in Example 8;

FIG. 82 is a graph illustrating cell growth as a function of the concentration of the 560 compound, in the MM.1s cell proliferation assay described in Example 8;

FIG. 83 is a graph illustrating cell growth as a function of the concentration of the 560 compound, in the MOLP-8 cell proliferation assay described in Example 8;

FIG. 84 is a graph illustrating cell growth as a function of the concentration of the 560 compound, in the Jeko-1 Parental cell proliferation assay described in Example 8;

FIG. 85 is a graph illustrating cell growth as a function of the concentration of the 560 compound, in the Jeko-1 Lenalidomine Resistant cell proliferation assay described in Example 8;

FIG. 86 is a graph illustrating cell growth as a function of the concentration of the 560 compound, in the Jeko-1 Bortezomib Resistant cell proliferation assay described in Example 8;

FIG. 87 is a graph illustrating cell growth as a function of the concentration of the 561 compound, in the MIA PaCa-2 cell proliferation assay described in Example 8;

FIG. 88 is a graph illustrating cell growth as a function of the concentration of the 561 compound, in the ASPC-1 cell proliferation assay described in Example 8;

FIG. 89 is a graph illustrating cell growth as a function of the concentration of the 561 compound, in the BxPC-3 cell proliferation assay described in Example 8;

FIG. 90 is a graph illustrating cell growth as a function of the concentration of the 561 compound, in the AN3CA cell proliferation assay described in Example 8;

FIG. 91 is a graph illustrating cell growth as a function of the concentration of the 561 compound, in the HEC-1a cell proliferation assay described in Example 8;

FIG. 92 is a graph illustrating cell growth as a function of the concentration of the 561 compound, in the MDA-MB-231 cell proliferation assay described in Example 8;

FIG. 93 is a graph illustrating cell growth as a function of the concentration of the 561 compound, in the MDA-MB-468 cell proliferation assay described in Example 8;

FIG. 94 is a graph illustrating cell growth as a function of the concentration of the 561 compound, in the HCC70 cell proliferation assay described in Example 8;

FIG. 95 is a graph illustrating cell growth as a function of the concentration of the 561 compound, in the H1975 cell proliferation assay described in Example 8;

FIG. 96 is a graph illustrating cell growth as a function of the concentration of the 561 compound, in the H1650 cell proliferation assay described in Example 8;

FIG. 97 is a graph illustrating cell growth as a function of the concentration of the 561 compound, in the A2780 cell proliferation assay described in Example 8;

FIG. 98 is a graph illustrating cell growth as a function of the concentration of the 561 compound, in the A2780CP cell proliferation assay described in Example 8;

FIG. 99 is a graph illustrating cell growth as a function of the concentration of the 561 compound, in the RXF-393 cell proliferation assay described in Example 8;

FIG. 100 is a graph illustrating cell growth as a function of the concentration of the 561 compound, in the A498 cell proliferation assay described in Example 8;

FIG. 101 is a graph illustrating cell growth as a function of the concentration of the 561 compound, in the N87 cell proliferation assay described in Example 8;

FIG. 102 is a graph illustrating cell growth as a function of the concentration of the 561 compound, in the SiHA cell proliferation assay described in Example 8;

FIG. 103 is a graph illustrating cell growth as a function of the concentration of the 561 compound, in the FaDu cell proliferation assay described in Example 8;

FIG. 104 is a graph illustrating cell growth as a function of the concentration of the 561 compound, in the DOHH-2 cell proliferation assay described in Example 8;

FIG. 105 is a graph illustrating cell growth as a function of the concentration of the 561 compound, in the SU-DHL-4 cell proliferation assay described in Example 8;

FIG. 106 is a graph illustrating cell growth as a function of the concentration of the 561 compound, in the OCI-LY3 cell proliferation assay described in Example 8;

FIG. 107 is a graph illustrating cell growth as a function of the concentration of the 561 compound, in the JIM1 cell proliferation assay described in Example 8;

FIG. 108 is a graph illustrating cell growth as a function of the concentration of the 561 compound, in the KMM-1 cell proliferation assay described in Example 8;

FIG. 109 is a graph illustrating cell growth as a function of the concentration of the 561 compound, in the KMS-11 cell proliferation assay described in Example 8;

FIG. 110 is a graph illustrating cell growth as a function of the concentration of the 561 compound, in the KMS-27 cell proliferation assay described in Example 8;

FIG. 111 is a graph illustrating cell growth as a function of the concentration of the 561 compound, in the KMS-34 cell proliferation assay described in Example 8;

FIG. 112 is a graph illustrating cell growth as a function of the concentration of the 561 compound, in the H929 cell proliferation assay described in Example 8;

FIG. 113 is a graph illustrating cell growth as a function of the concentration of the 561 compound, in the L363 cell proliferation assay described in Example 8;

FIG. 114 is a graph illustrating cell growth as a function of the concentration of the 561 compound, in the MM.1s cell proliferation assay described in Example 8;

FIG. 115 is a graph illustrating cell growth as a function of the concentration of the 561 compound, in the MOLP-8 cell proliferation assay described in Example 8;

FIG. 116 is a graph illustrating cell growth as a function of the concentration of the 561 compound, in the Jeko-1 Parental cell proliferation assay described in Example 8;

FIG. 117 is a graph illustrating cell growth as a function of the concentration of the 561 compound, in the Jeko-1 Lenalidomine Resistant cell proliferation assay described in Example 8;

FIG. 118 is a graph illustrating cell growth as a function of the concentration of the 561 compound, in the Jeko-1 Bortezomib Resistant cell proliferation assay described in Example 8;

FIG. 119 is a graph illustrating cell growth as a function of the concentration of the confirmed 560 compound, in the S2-007 pancreatic ductal adenocarcinoma cell proliferation assay described in Example 15;

FIG. 120 is a graph illustrating cell growth as a function of the concentration of the confirmed 560 compound, in the MiaPaCa-2 pancreatic ductal adenocarcinoma cell proliferation assay described in Example 15;

FIG. 121 is a graph illustrating cell growth as a function of the concentration of the confirmed 562 compound, in the S2-007 pancreatic ductal adenocarcinoma cell proliferation assay described in Example 15;

FIG. 122 is a graph illustrating cell growth as a function of the concentration of the confirmed 562 compound, in the MiaPaCa-2 pancreatic ductal adenocarcinoma cell proliferation assay described in Example 15;

FIG. 123 is a series of photographs depicting the colony formations as a function of the concentration of the confirmed 560 compound, in the S2-007 pancreatic ductal adenocarcinoma cell colony formation assay described in Example 16;

FIG. 124 is a series of photographs depicting the colony formations as a function of the concentration of the confirmed 560 compound, in the MiaPaCa-2 pancreatic ductal adenocarcinoma cell colony formation assay described in Example 16;

FIG. 125 is a series of photographs depicting the colony formations as a function of the concentration of the confirmed 562 compound, in the S2-007 pancreatic ductal adenocarcinoma cell colony formation assay described in Example 16;

FIG. 126 is a series of photographs depicting the colony formations as a function of the concentration of the confirmed 562 compound, in the MiaPaCa-2 pancreatic ductal adenocarcinoma cell colony formation assay described in Example 16;

FIG. 127 is a further series of photographs depicting the colony formations as a function of the concentration of the confirmed 562 compound, in the MiaPaCa-2 pancreatic ductal adenocarcinoma cell colony formation assay described in Example 16;

FIG. 128 is a set of bar graphs illustrating the results of a cell cycle assay using the confirmed 560 compound with S2-007 cells over 24 and 48 hours, as described in Example 17, with Sub G0;

FIG. 128A is a set of bar graphs illustrating the results of a cell cycle assay using the confirmed 560 compound with S2-007 cells over 24 and 48 hours, as described in Example 17, without Sub G0;

FIG. 129 is a set of bar graphs illustrating the results of a cell cycle assay using the confirmed 560 compound with MiaPaCa-2 cells over 24, 48, and 72 hours, as described in Example 17, with Sub G0;

FIG. 129A is a set of bar graphs illustrating the results of a cell cycle assay using the confirmed 560 compound with MiaPaCa-2 cells over 24, 48, and 72 hours, as described in Example 17, without Sub G0;

FIG. 130 is a set of bar graphs illustrating the results of a cell cycle assay using the confirmed 562 compound with S2-007 cells over 24, 48, and 72 hours, as described in Example 17, with Sub G0;

FIG. 130A is a set of bar graphs illustrating the results of a cell cycle assay using the confirmed 562 compound with S2-007 cells over 24, 48, and 72 hours, as described in Example 17, without Sub G0;

FIG. 131 is a set of bar graphs illustrating the results of a cell cycle assay using the confirmed 562 compound with MiaPaCa-2 cells over 24, 48, and 72 hours, as described in Example 17, with Sub G0; and

FIG. 131A is a set of bar graphs illustrating the results of a cell cycle assay using the confirmed 562 compound with MiaPaCa-2 cells over 24, 48, and 72 hours, as described in Example 17, without Sub G0.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The therapeutic agents of the invention are used in therapeutically effective amounts, i.e., amounts that will elicit the biological or medical response of a tissue, system, or subject that is being sought, and in particular to elicit some desired therapeutic effect against a variety of human diseases, and especially cancers; in the case of cancers, the agents operate by preventing and/or inhibiting proliferation and/or survival of cancerous cells, including cancer stem cells, and/or by slowing the progression of cancers. Those skilled in the art recognize that an amount may be considered therapeutically effective even if the condition is not totally eradicated or prevented, but it or its symptoms and/or effects are improved or alleviated partially in the subject. Of course, the appropriate makeup of the agents hereof and dosing regimens using such agents will depend on the particular cancer being treated, the extent of the disease, and other factors related to the patient as determined by those skilled in the art. Hence, the terms “therapeutic” or “treat,” as used herein, refer to products or processes in accordance with the invention that are intended to produce a beneficial change in an existing condition (e.g., cancerous tissue, tumor size, metastases, etc.) of a subject, such as by reducing the severity of the clinical symptoms and/or effects of the condition, and/or reducing the duration of the symptoms/effects of a subject.

Additional ingredients may be included with the chemotherapeutic agents of the invention for administration to the subject. Such additional ingredients include, other active agents, preservatives, buffering agents, salts, carriers, excipients, diluents, or other pharmaceutically acceptable ingredients. The active agents that could be included in the compositions include antiviral, antibiotic, or other anticancer compounds; the latter could include the compounds described in PCT application serial number PCT/US2015/055968, such as curcumin, harmine, and isovanillin, and metabolites, dimers, derivatives, isomers, enantiomers (both D and L), tautomers, esters, complexes and salts of any of the foregoing.

The therapeutic agents of the invention give significant and unexpected therapeutic results, particularly in the context of anti-cancer results. In use, a therapeutically effective amount of an agent or composition in accordance with the invention is administered to a subject in need thereof. Such may comprise a single unit dosage or, more usually, periodic (e.g., daily) administration of lower dosages over time.

The dosages may be administered in any convenient manner, such as by oral, rectal, nasal, ophthalmic, parenteral (including intraperitoneal, gastrointestinal, intrathecal, intravenous, cutaneous (e.g., dermal patch), subcutaneous (e.g., injection or implant), or intramuscular) administrations. The dosage forms of the invention may be in the form of liquids, gels, suspensions, solutions, or solids (e.g., tablets, pills, or capsules). Moreover, therapeutically effective amounts of the agents of the invention may be co-administered with other chemotherapeutic agent(s), where the two products are administered substantially simultaneously or in any sequential manner.

Levels of dosing using the compositions of the invention are quite variable owing to factors such as the patient's age, patient's physical condition, weight, the type of condition(s) being treated (e.g., specific cancer(s)), and the severity of the conditions. In general, however, regardless of the dosage form or route of administration employed, such as liquid solutions or suspensions, capsules, pills, or tablets, via oral, parenteral, or injection, the compositions should be dosed of from about 5 to 2000 mg per day, and more usually from about 100-800 mg per day. Such dosages may be based on a single administration per day, but more usually multiple administrations per day.

Additional advantages of the various embodiments of the invention will be apparent to those skilled in the art upon review of the disclosure herein and the working examples below. It will be appreciated that the various embodiments described herein are not necessarily mutually exclusive unless otherwise indicated herein. For example, a feature described or depicted in one embodiment may also be included in other embodiments, but is not necessarily included. Thus, the present invention encompasses a variety of combinations and/or integrations of the specific embodiments described herein.

As used herein, the phrase “and/or,” when used in a list of two or more items, means that any one of the listed items can be employed by itself or any combination of two or more of the listed items can be employed. For example, if a composition is described as containing or excluding components A, B, and/or C, the composition can contain or exclude A alone; B alone; C alone; A and B in combination; A and C in combination; B and C in combination; or A, B, and C in combination.

The present description also uses numerical ranges to quantify certain parameters relating to various embodiments of the invention. It should be understood that when numerical ranges are provided, such ranges are to be construed as providing literal support for claim limitations that only recite the lower value of the range as well as claim limitations that only recite the upper value of the range. For example, a disclosed numerical range of about 10 to about 100 provides literal support for a claim reciting “greater than about 10” (with no upper bounds) and a claim reciting “less than about 100” (with no lower bounds).

As used herein, pharmaceutically acceptable salts with reference to the therapeutic compounds of the present invention mean salts of the compounds which are pharmaceutically acceptable, i.e., salts which are useful in preparing pharmaceutical compositions that are generally safe, non-toxic, and neither biologically nor otherwise undesirable and are acceptable for human pharmaceutical use, and which possess the desired degree of pharmacological activity. Such pharmaceutically acceptable salts include acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or with organic acids such as 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, 2-naphthalenesulfonic acid, 3-phenylpropionic acid, 4,4′-methylenebis(3-hydroxy-2-ene-1-carboxylic acid), 4-methylbicyclo[2.2.2]oct-2-ene-1-carboxylic acid, acetic acid, aliphatic mono- and dicarboxylic acids, aliphatic sulfuric acids, aromatic sulfuric acids, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, carbonic acid, cinnamic acid, citric acid, cyclopentanepropionic acid, ethanesulfonic acid, fumaric acid, glucametacin acid, gluconic acid, glutamic acid, glycolic acid, heptanoic acid, hexanoic acid, hydroxynaphthoic acid, lactic acid, laurylsulfuric acid, maleic acid, malic acid, malonic acid, Mandela acid, methanesulfonic acid, muconic acid, o-(4-hydroxybenzoyl)benzoic acid, oxalic acid, p-chlorobenzenesulfonic acid, phenyl-substituted alkanoic acids, propionic acid, p-toluenesulfonic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, tartaric acid, tertiarybutyl acetic acid, trimethylacetic acid, and the like. Pharmaceutically acceptable salts also include base addition salts which may be formed when acidic protons present are capable of reacting with inorganic or organic bases. Acceptable inorganic bases include sodium hydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide and calcium hydroxide. Acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine and the like. It should be recognized that the particular anion or cation forming a part of any salt of this invention is not critical, so long as the salt, as a whole, is pharmacologically acceptable. Additional examples of pharmaceutically acceptable salts and their methods of preparation and use are presented in Handbook of Pharmaceutical Salts Properties, and Use, P. H. Stahl & C. G. Wermuth eds., ISBN 978-3-90639-058-1 (2008).

In preparing the compounds of the invention, use should be made of starting ingredients of relatively high purity, typically at least about 90% by weight pure, and more preferably at least about 98% by weight pure. The use of naturally occurring sources for the ingredients is generally not appropriate or desirable, because these naturally occurring products may contain relatively small amounts of the desired components and/or have potentially interfering compounds therein. Moreover, use of low-purity ingredients often leads to little or no compounds in accordance with the invention.

Thus, the preferred starting compounds or components of the invention are either synthetically derived or derived from one or more naturally occurring product(s) which have been significantly modified so as to contain at least about 90% by weight (more preferably at least about 98% by weight) of the desired component. As used herein, “synthetically derived” means that the component in question was synthesized using specific starting ingredients and one or more chemical and/or biological reactions to obtain substantially pure compounds. Modification of naturally occurring products may involve extractions, or any other physical or chemical steps to achieve the desired end product.

As used herein, the terms “alkyl,” “alkenyl,” “alkynyl,” mean and are intended to cover straight, branched chain, and cyclic groups. “Amines” means and is intended to cover primary, secondary, and tertiary amines. “Sulfur groups” means and is intended to cover thiols, sulfides, disulfides, and sulfoxides. “Derivative” means and is intended to cover compounds, moieties, and/or groups which are substituted with atoms, groups, or side chains which do not materially degrade (e.g., no more than about 20%, preferably no more than about 10%, degradation) of the performance of the compound, moiety, or group as compared with the unsubstituted versions thereof.

As indicated, certain preferred compounds or agents of the invention comprise a pair of fused polycyclic moieties, each including an N-containing ring, where the fused polycyclic moieties are bound or linked by a single tether or linker moiety, which are schematically illustrated as

PCM1—L—PCM2

where PCM1 and PCM2 are the fused polycyclic moieties (which may be the same or different), where L is the tether or linker. As indicated by this schematic, the linker L may be attached to PCM1 and PCM2 at any position on any ring thereof, and the bonding sites need not be the same for both PCM1 and PCM2. The fused polycyclic and linker moieties are described below.

The Fused Polycyclic Compounds or Moieties

The fused polycyclic moieties of the invention are derived or synthesized from starting ingredients which yield compounds or moieties having the following generalized structure:

where one of the terminal rings is a 6-membered ring including at least one N heteroatom at any valence-permitted position(s) around the 6-membered ring (the single N-atom illustrated in Structure I is exemplary only, both in terms of the position of the N-atom, and the number of N-atoms). This 6-membered ring may be aryl in character (e.g., a pyrido ring), or non-aryl (e.g., a piperidine ring), or contain multiple N-atoms (e.g., a piperazine ring). Further with respect to Structure I, RG1 is fused with the terminal 6-membered N-atom-containing ring and has from 5-8 atoms (as used herein, “fused” refers to the fact that the fused rings share 2 adjacent atoms or, in other words, 1 covalent bond). Ring RG2 may be nothing (i.e., the moiety is bicyclic) and, if present, ring RG2 is fused with ring RG1 and with the terminal six-membered ring, and has from 5-8 atoms. In both RG1, RG2 (where present), and the six-membered N-containing ring, the majority of ring atoms in each case are carbon atoms. However, these rings may also include one or more heteroatoms, such as S, O, or N.

The interior dotted lines illustrated in RG1 and RG2 represent the fact that the individual rings may have one or more double bonds and may be aryl or non-aryl in character. As indicated, in the six-member N-atom-containing ring, the N-atom(s) may be at any permitted position around the ring, and the dotted lines represent that the six-membered ring may have 1, 2, or 3 double bonds. The n subscript on each Y1 represents the fact that there may be single or multiple substituents at any permitted position(s) around the six-membered N-atom-containing ring, RG1, and/or RG2; preferably, each n is independently either 1, 2, or 3. Each Y1 and Y2 is independently selected from the group consisting of nothing, OH, C1-C12 (preferably C1-C₄) alkyl, alkenyl, and alkynyl groups, C1-C12 (preferably C1-C4) alkoxy and alkoxyphenyl groups, aryl and aryloxy groups, aldehyde and carbaldehyde groups, amines, nitro groups, nitrile groups, C2-C6 carboxylic acid groups, boronic groups, sulfur groups, and amino acids, where any of the aforementioned may be substituted with N, S, O, B, or halogen atoms.

Exemplary bicyclic compounds corresponding to the moieties in accordance with Structure I may include quinoline and derivatives thereof, purine and derivatives thereof, as well as quinolin-2-amine, 6-bromo-2-methylquinoline, 2-hydroxy-4-methylquinoline, 4-chloro-7-methoxyquinoline, 8-quinolineboronic acid, quinoxaline, 8-aminoisoquinoline, 5-chloro-3-methylbenzothiothene, 4-nitroquinoline-N-oxide, 1-methylisoquinoline, 7-methylquinoline, 6,7-dimethoxyquinazoline-2,4-dione, 6-chloroquinoline, 1-chloroisoquinoline, 4-chloroquinoline, 8-chloroquinoline, isocarbostyril, 8-hydroxyquinoline-5-sulfonic acid, isoquinoline N-oxlide, 6-fluoroquinaldine, 2-chloroquinoline-3-carbaldehyde, 5-nitroisoquinoline, 2,6-dimethylquinoline, 3-hydroxyquinoline, 2-methyl-6-quinolinecarboxylic acid, 6-bromoisoquinoline, 8-mercaptoquinoline hydrochloride, quinoline-4-carboxylic acid, 6-bromoquinoline, 7-bromoquinoline, 6-nitroquinoline, decahydroquinoline, 4-hydroxyquinoline, 8-methylquinoline, 3-hydroxy-2-methyl-4-quinolinecarboxylic acid, 6-quinolinecarboxylic acid, 3-quinolinecarboxylic acid, 2-hydroxy-4-quinolinecarboxylic acid, 2,4-dimethylquinoline, 1-isoquinolinecarbonitrile, 7-chloro-2-methylquinoline, 1-methyl-3,4-dihydroisoquinoline, 4-methyl-6H,7H-thieno[3,2-c]pyridine, 7-methyl-4H,5H-thieno[2,3-c]pyridine, 1-ethyl-3,4-dihydroisoquinoline, 5-methyl-7,8-dihydro-1,6-naphthyridine, 1,4-dimethyl-3,4-dihydro-2,7-naphthyridine, 5-methyl-7,8-dihydro-1,6-naphthyridine, and 4-methyl-6,7-dihydrothienol[3,2-c]pyridine.

A variety of fused tricyclic compounds corresponding to the moieties in accordance with Structure I are also useful in the invention. One class of fused tricyclic compounds are O-carboline compounds and derivatives thereof, having a tricyclic ring system, e.g., a bicycle made up of a six-membered benzene ring and a fused five-membered pyrrole ring, with a terminal N-containing ring fused with the intermediate pyrrole ring. Exemplary O-carbolines include tryptoline, pinoline, harmane, harmine, harmaline, tetrahydroharmaline, and 9-methyl-β-carboline. Harmaline is in some instances preferred for use in the invention.

Harmaline (7-methoxy-1-1-methyl-4,9-dihydro-3H-pyrido[3,4-b]indole) is a fluorescent psychoactive alkaloid from the group of harmala alkaloids and O-carbolines, and occurs in various plants, such as Peganum harmala. Harmaline is identified as CAS #304-21-2, and exists in two tautomeric forms:

As used herein, “harmaline” refers to either or both tautomers. Other harmaline components are described below.

Some harmaline components have the structure

where the numbered 6-member fused ring is a N-heterocycle with a single N atom at any of the positions 2-5, and the R6′ substituents may be located at any ring position; R5′ is H, OH, C1-C12 (preferably C1-C₄) alkoxy, aryloxy (e.g., benzyloxy or phenoxy), carboxy, biphenyl, nitro, carboxylate; and R6′ is H, OH, C1-C12 (preferably C1-C₄) alkyls, or a C1-C12 (preferably C1-C4) carboxylic acid.

Representative compounds of this type include harmaline and the following:

In the foregoing representative compounds, any methoxy substituent may be replaced by a C2-C4 alkoxy group, or by a phenoxy group.

The Linker Compounds and Moieties

Each linker L provides two bonding branches from a single atom forming at least a part of the linker moiety. Thus, the linker moiety may present an effective “V” or “Y” configuration, with the single atom at the lower vertex (as depicted in Structure II below) with the two bonding branches respectively bonded to the fused polycyclic moieties. Thus, a linker may be a single methylene group (CH2), where the fused polycyclic moieties are bonded to the carbon atom of the methylene group to present an effective “V” configuration. In like manner, the linker may comprise a pair of alkyl groups with an intermediate carbon atom such as CH3-C—CH3, so that the fused polycyclic moieties are bonded to the intermediate carbon atom. Thus, preferred linkers include multiple atoms, one of which is the bonding atom for the fused polycyclic moieties. The single bonding atom of the linkers may be selected from non-metals, and especially atoms of carbon, nitrogen, oxygen, fluorine, phosphorous, sulfur, chlorine, bromine, and iodine. Metal atoms, such as Pt, are normally less preferred. In addition, the bonding between the single atom and the bonding branches may be classical covalent bonding, meaning that each atom participating in the bonding contributes at least one electron as a part of a molecular orbital. However, typical metal bonding, such as coordinate or dative bonding, is generally less favored.

It will be appreciated that linker compounds or moieties serve to separate the fused polycyclic moieties forming a part of the compounds of the invention and may also contribute to the morphology and/or steric characteristics of the complete compounds. As used herein, and in keeping with conventional linker nomenclature, the entirety of any multiple-atom linker moiety between the fused polycyclic moieties is considered to be the “linker,” without any artificial separation of such a multiple-atom linker moiety, where one atom of the linker moiety is deemed to be the “linker” and with the remainder of the linker moiety not being considered as a part of the “linker.” For example, if a propyl moiety is used as the linker moiety where two fused polycyclic moieties are bonded to respectively bonded to the terminal carbons of the propyl moiety, it would be inappropriate and not in keeping with the invention to deem one of the terminal CH2 to methylene groups as the linker, while disregarding the presence of the remaining CH2-CH2 group as a part of the linker.

In certain preferred compounds of the invention, the fused tricyclic moieties are bonded to the linker through a single atom, and wherein this single atom is the carbon atom of a methine group. A “methine group” is defined by the Illustrated Glossary of Organic Chemistry as a portion of a molecular structure equivalent to methane minus three hydrogen atoms, i.e., a CH group. A methine group is to be contrasted with a “methylene group” defined as a portion of a molecular structure equivalent to methane minus two hydrogen atoms, or a CH2 group.

Some linker moieties may be derived from aldehydes, where both of the fused polycyclic moieties are bonded to the carbonyl carbon of the aldehyde functional group, thereby presenting an effective “Y” configuration. In certain embodiments, appropriate aldehyde linker moieties are characterized by a six-membered ring with an attached aldehyde functional group, of the following structure.

where the substituents may be located at any position around the ring. R1′ is a C1-C12 (preferably C1-C₄) aldehyde, R2′-R5′ are independently and selectively taken from the group consisting of H, OH, C1-C12 (preferably C1-C4) alkyl groups, C2-C12 (preferably C2-C5) alkenyl groups, C1-C12 (preferably C1-C4) alkoxy groups, C1-C12 (preferably C1-C4) aldehyde groups, acetate, isobutyrate, phenyl, phenoxy, benzyloxy, C2-C12 (preferably C2-C₆) alkyl esters, halo (e.g., F, Br, I, Cl), primary and secondary amines, nitro, and mixtures thereof. The dotted bond lines in the six-membered ring represent that the six-membered ring may be cyclohexane, or have one, two, or three carbon-carbon double bonds (e.g., cyclohexene, cyclohexadiene, phenyl, or derivatives thereof).

Representative compounds of this type include vanillin, benzaldehyde, cinnamaldehyde, cuminaldehyde, orthovanillin, perillaldehyde, cyclohexanecarboxaldehyde, and the following:

Still further phenyl aldehydes useful in the invention as linkers include moieties of 2-methoxybenzaldehyde, 3-ethoxy-4-hydroxybenzaldehyde, 4-formyl-2-methoxyphenyl isobutyrate, 3,4-dimethoxybenzaldehyde, 4-hydroxy-3-methoxy-5-nitrobenzaldehyde, 4-formyl-2-methoxyphenyl acetate, 3-hydroxy-5-methoxybenzaldehyde, 2-hydroxy-4-methoxybenzaldehyde, 3-chloro-4-hydroxy-5-methoxybenzaldehyde, 4-(benzyloxy)-3-methoxybenzaldehyde, 3-hydroxy-4,5-dimethoxybenzaldehyde, 3-bromo-4-hydroxy-5-methoxybenzaldehyde, 2-bromo-3-hydroxy-4-methoxybenzaldehyde, 3-hydroxy-2-iodo-4-methoxybenzaldehyde, 3-methoxybenzaldehyde, 3-phenoxybenzaldehyde, 4-phenoxybenzaldehyde, [1,1′-biphenyl]-3-carbaldehyde, 4-fluoro-3-phenoxybenzaldehyde, 3-fluorobenzaldehyde, 4-fluorobenzaldehyde, 3,5-difluorobenzaldehyde, 2,4,5-trifluorobenzaldehyde, 2,3,4,5,6-pentafluorobenzaldehyde, 4-methylbenzaldehyde, terephthalaldehyde, 4-chlorobenzaldehyde, 4-(prop-1-en-2-yl)cyclohex-1-ene-1-carbaldehyde, 4-isopropylbenzaldehyde, and cyclohexanecarbaldehyde.

In other embodiments, aliphatic or alkenyl aldehydes may be used as linkers. Generally, such linkers are moieties of aldehydes, such as C1-C12 alkyl or C2-C12 alkenyl aldehydes, and include representative compounds such as (E)-hex-2-enal (C6H10O, Exact Mass: 98.07), 3-methylbutanal (isovaleraldehyde) (C5H10O, Exact Mass: 86.07), 3,7-dimethyloct-6-enal (citronellal) (C10H18O, Exact Mass: 154.14), 7-hydroxy-3,7-dimethyloctanal (hydroxycitronellal) (C10H20O2, Exact Mass: 172.15), and dodecyl aldehyde (C12H240, Exact Mass: 184.18).

The Complete Compounds of the Invention

As noted previously, one generalized form of the compounds is set forth in the schematic representation

PCM1-L—PCM2.

Preferred species of this representation are set forth in the following Structure II

where it will be seen that the intermediate tether or linker L is bonded to the six-membered, N-containing ring of the respective fused polycyclic moieties of Structure I, and specifically at a single atom forming a lower vertex and at least a part of the linker L. The bonding sites of the linker L to the fused polycyclic moieties may be at any permitted locations around the six-membered rings, including at the N-heteroatom (in which case Y2 would be nothing), and such bonding sites need not be the same for the respective polycyclic moieties. The six-membered terminal N-containing ring, RG1, RG2, the Y1 and Y2 substituents, and the n values, are those previously defined with respect to Structure I.

One class of compounds (Structure III below) has a central linker bonded to the N-containing B rings at respective positions at an ortho carbon atom relative to the nitrogen atom, where each of the β-carboline groups may independently be substituted or unsubstituted. “Substituted” with respect to the first moiety ring, and the β-carboline groups, means that these may be substituted at any position (and independently in the case of the respective β-carboline groups) with any substituent which does not materially degrade (e.g., no more than about 20%, preferably no more than about 10%, degradation) of the performance of the compound as compared with the unsubstituted version thereof More particularly, certain other compounds of this type have the generic Structure III:

wherein each of X1, X2, X3, and X9, is independently selected from the group consisting of nothing, OH, C1-C12 alkyl, alkenyl, and alkynyl groups, C1-C12 alkoxy and alkoxyphenyl groups, aryl and aryloxy groups, aldehyde and carbaldehyde groups, amines, nitro groups, nitrile groups, C2-C6 carboxylic acid groups, boronic groups, sulfur groups, and amino acids, where any of the aforementioned may be substituted with N, S, O, B, or halogen atoms, Z contains the single bonding atom described above and is selected from the group consisting of C1-C12 alkyl, alkenyl, and alkynyl groups, C1-C12 alkoxy and alkoxyphenyl groups, aryl and aryloxy groups, aldehyde and carbaldehyde groups, amines, nitro groups, nitrile groups, C2-C6 carboxylic acid groups, boronic groups, sulfur groups, and amino acids, where any of the aforementioned may be substituted with N, S, O, B, or halogen atoms. Each X3 may be attached at any position around the corresponding terminal phenyl moieties of the β-carboline groups. Each Y is independently nothing (e.g., there is a direct bond between the two B rings, or Z may be directly coupled to one or both of the B rings), H, OH, C1-C12 (preferably C1-C4) alkyl, alkenyl, and alkynyl groups, C1-C12 (preferably C1-C4) alkoxy and alkoxyphenyl groups, aryl and aryloxy groups, aldehyde groups, amines, nitro groups, nitrile groups, C2-C6 carboxylic acid groups, boronic groups, sulfur groups, and amino acids, where any of the aforementioned may be substituted with N, S, O, B, or halogen atoms. Preferably, Y is a C1-C12 (preferably C1-C4) group composed of C, CH, and/or CH2 atoms or groups, and Z is C, CH, or CH2. X9 is preferably selected from the group consisting of nothing (e.g., M may be bonded to Z), C1-C12 (preferably C1-C4) alkyl groups, and C2-C12 (preferably C2-05) alkenyl groups. M is selected from the group consisting of Structure IIIA, nothing, OH, C1-C12 (preferably C1-C4) alkyl, alkenyl, and alkynyl groups, C1-C12 (preferably C1-C4) alkoxy and alkoxyphenyl groups, aryl and aryloxy groups, aldehyde groups, amines, nitro groups, nitrile groups, C2-C6 carboxylic acid groups, boronic groups, sulfur groups, and amino acids, where any of the aforementioned may be substituted with N, S, O, B, or halogen atoms. Each X4, X5, X6, X7, and X8 of Structure IIIA is attached at any position around the A ring and is independently selected from the group consisting of nothing, OH, C1-C12 (preferably C1-C4) alkyl, alkenyl, and alkynyl groups, C1-C12 (preferably C1-C4) alkoxy and alkoxyphenyl groups, aryl and aryloxy groups, aldehyde groups, amines, nitro groups, nitrile groups, C2-C6 carboxylic acid groups, boronic groups, sulfur groups, and amino acids, where any of the aforementioned may be substituted with N, S, O, B, or halogen atoms. The designation

in the A ring refers to the fact that there may optionally be 0, 1, 2, or 3 double bonds (e.g., the A ring may be cyclohexane, cyclohexene, cyclohexadiene, benzene, or derivatives thereof), and wherein the designation

in connection with the two B rings refers to the fact that there may optionally be: 1) one or two non-fused double bonds at one or two valence-permitted positions around either or both of the B rings, such as illustrations a-d below; 2) a double bond between either or both of the B rings and Y or Z, with or without an additional non-fused double bond at any valence-permitted position around the corresponding N-containing ring, such as illustrations e-g below. In instances of 2) where there is a double bond between the nitrogen atom of either N-containing ring and Y or Z, X1 may be nothing, such as illustrations a-c and g. However, if there is no such nitrogen double bond, the corresponding X1 is as defined above, and is preferably selected from the group consisting of H, OH, and C1-C12 (preferably C1-C4) alkyl groups, such as illustrations d-f; or 3) either or both of the B rings are free of non-fused double bonds, and each X1 is as set forth above, and is preferably from the group consisting of H, OH, and C1-C12 (more preferably C1-C4) alkyl groups.

In preferred instances where Z is a methine CH group, X9 is not nothing or H.

Set forth below are illustrations depicting certain exemplary double bond configurations of either or both of the B rings of the above Structure III.

Advantageously, each X1 is nothing, each X2 is H, and each X3 is methoxy.

In certain embodiments of Structure III, M is the 1A ring, both of X1 are nothing, both of X3 are methoxy, 2 of X4, X5, X6, X7, and X8 are H, at least one of X4, X5, X6, X7, and X8 is selected from the group consisting of H, —OH, methoxy, ethoxy, phenoxy, C2-C5 alkenyl groups, F, and C1, with the provisos that: 1) when one or more of X4, X5, X6, X7, and X8 is/are F or C1, the remainder of X4, X5, X6, X7, and X8 are all H; 2) only one of X4, X5, X6, X7, and X8 may be phenoxy, and in such case, the remainder of X4, X5, X6, X7, and X8 are all H.

In other embodiments, certain compounds containing two harmaline moieties and a single phenyl moiety derived from a phenyl aldehyde compound are provided, having the general Structure IV:

where X10 is —CH═CH—, X11, X12, X13, and X14 are each independently selected from the group consisting of H, —OH, methoxy, ethoxy, and phenoxy, F, and C1, with the provisos that: 1) at least one of X12, X13, or X14 is H; 2) when one or more of X11, X12, X13, or X14 is/are F or C1, the remainder of the X11, X12, X13, and X14 are all H; 3) a phenoxy group is present only at X12, and X11, X13, and X14 are all H, and 4) if a methoxy or ethoxy is present, at least one such methoxy or ethoxy must be at either the 2 or 3 position, wherein the designation

in connection with the two N-containing rings refers to the fact that there may optionally be: 1) one or two non-fused double bonds at one or two valence-permitted positions around either or both of the N-containing rings, such as illustrations a-c below; 2) a double bond between either or both of the N-containing rings and the adjacent carbon atoms, with or without an additional non-fused double bond at any valence-permitted position around the corresponding N-containing ring, such as illustration g below; or 3) either or both of the N-containing rings are free of non-fused double bonds, and each X1 is independently selected from the group consisting of H, OH, and C1-C12 (preferably C1-C4) alkyl groups.

Where X10 is nothing and X11, X12, X13, and X14 are all H, the resultant structure is the 560 compound described in Example 12; where X10 is nothing, X11 is H, X12 is methoxy, X13 is —OH, and X14 is H, the resultant structure is the 562 compound described in Example 14; where X10 is nothing, X11 is —OH, X12 is methoxy, and X13 and X14 are both H, the resultant structure is the principal 523 compound described below; where X10 is nothing, X11, X13, and X14 are all H, and X12 is phenoxy, the resultant structure is the 594 compound described in Example 24; and where X10 is —CH═CH—, and all of X11, X12, X13, and X14 are H, the resultant structure is the 561 diharmaline compound set forth hereinafter as the primary compound of harmaline and cinnamaldehyde.

In other embodiments, certain compounds within the ambit of Structure IV containing two harmaline moieties and a single linker moiety are provided. These compounds are selected from the group consisting of

Synthesis of the Complete Compounds of the Invention

In preparing the compounds of the invention, use should be made of starting ingredients of relatively high purity, typically at least about 90% by weight pure, and more preferably at least about 98% by weight pure. The use of naturally occurring sources for the ingredients is generally not appropriate or desirable, because these naturally occurring products may contain relatively small amounts of the desired components and/or have potentially interfering compounds therein. Moreover, use of low-purity ingredients often leads to little or no compounds in accordance with the invention.

Thus, the preferred starting compounds or components of the invention are either synthetically derived or derived from one or more naturally occurring product(s) which have been significantly modified so as to contain at least about 90% by weight (more preferably at least about 98% by weight) of the desired component. As used herein, “synthetically derived” means that the component in question was synthesized using specific starting ingredients and one or more chemical and/or biological reactions to obtain substantially pure compounds. Modification of naturally occurring products may involve extractions, or any other physical or chemical steps to achieve the desired end product.

One method of preparing the compounds of the invention, particularly where the linker moiety is derived from an aldehyde, involves the direct reaction between the aldehyde and the fused polycyclic compounds of interest. Hence, products produced by this method are reaction products of an aldehyde and the fused polycyclic compounds.

In carrying out the aldehyde reactions between the aldehyde and fused polycyclic compound(s), of whatever types, the weight ratios of the aldehyde component(s) to the fused polycyclic compound(s) in the reaction mixtures should range from about 0.5:1 to 25:1, more preferably from about 0.7:1 to 6:1, and most preferably from about 1.5:1 to 4:1. In terms of weight amounts, the amounts of the aldehyde component(s) should range from about 25-95% by weight, and the weight of amount of the fused polycyclic compound(s) should be from about 5-75% by weight, with the total weight of these reactants taken as 100% by weight. In most cases, it is preferred that the weight amount of the aldehyde component(s) should be present in a weight excess relative to the amount of the fused polycyclic compound(s).

The components are usually mixed with an organic solvent, such as a C1-C4 lower alcohol (e.g., methanol, ethanol, or propanol) and/or dimethyl sulfoxide (DMSO), and allowed to stand for a period (typically from about 12 hours-4 weeks) at a temperature ranging from about 20-60° C. at ambient pressures. Alternately, the mixture may be refluxed (e.g., 30 minutes-2 hours at 50-85° C. in ethanol or 30 minutes at 55° C. in methanol). The reaction products can then be recovered in either liquid or solid form. Depending upon the selected solvent, the reaction products may exhibit different colors, but this does not affect the anti-cancer properties of the reaction products. Moreover, the particular reaction conditions are generally not critical.

The production of effective esters, metal complexes, and pharmaceutically acceptable salts of the compounds is quite straightforward and well within the skill of the art. For example, salts may be formed by reacting the products with inorganic or organic acids.

The reactants, reaction ratios, reactant amounts, and reaction conditions set forth above are suitable for all of the aldehyde reactions in accordance with the invention, and skilled artisans can readily determine the optimums through routine experimentation.

In some cases using the aldehyde reaction, it may be difficult to determine the precise structure(s) of the reaction products. However, molecular weights of the active reaction products can be determined, and such are important criteria for the active products. Thus, important reaction products of benzaldehyde and harmaline have a molecular weight of approximately 516, whereas such reaction products of vanillin and harmaline have a molecular weight of approximately 562. “Approximately” in connection with the molecular weights referred to herein means the listed molecular weights plus or minus 5 weight units. Also, the molecular weights of reaction product derivatives (e.g., reduction products produced by hydrogenation, esters, or salts) would be somewhat different; but such weights are easily calculated in light of the nature of the derivatives. Hence, the preferred molecular weights recited herein are for the non-derivatized versions of the reaction products.

A second synthesis method may be used when it is desired to produce fused tricyclic compounds such as β-carboline and its derivatives. Generally speaking, this method involves reacting an indole alkyl amine with a diacid to produce an intermediate, followed by a ring-closure reaction to produce the final compound of interest.

A third reaction method, particularly useful for the production of compounds having fused bicyclic moieties bonded with a linker moiety, is illustrated below.

Benzaldehyde/Harmaline 560 Compounds

Benzaldehyde is a benzene ring with an aldehyde substituent, and is the primary constituent of bitter almond oil. It is identified by CAS #100-52-7.

The aldehyde reaction between benzaldehyde and harmaline is preferably carried out by mixing together the two components at a weight ratio of about 2:1 (benzaldehyde:harmaline). Ethanol is then added to give a final reaction mixture concentration of about 10:1000 mg/mL, more preferably from about 700:1000 mg/mL to form a dispersion. The vial is then capped, and the mixture within the vial is allowed to stand in a warm water bath of about 50° C. (more broadly, about 40-60° C.) for about 3 days (more broadly, about 1-10 days). The solid compound is then washed with water and methanol, giving a final product of about 90-95% by weight purity.

Certain compounds are formed with one moiety of harmaline and one moiety of benzaldehyde, one of which has a molecular weight of approximately 320. Other products also have one moiety of harmaline and one of benzaldehyde, but have a molecular weight of approximately 302, owing to loss of water attendant to the reaction. These products are set forth below.

Further, useful compounds are formed with two harmaline moieties and a single linker moiety derived from benzaldehyde, with molecular weights of approximately 516, as follows.

Other compounds containing one moiety of benzaldehyde and two harmaline moieties include the following.

As explained in Example 12, a confirmed compound is

An analog of the above compound has a molecular weight of 520.68 and is a reduced version wherein the nitrogen atoms of the two harmaline moieties are hydrogenated, eliminating the double bond therein, as set forth below:

More broadly, however, appropriate benzaldehyde/harmaline compounds include one or more of the structure:

and the dimers, isomers, and tautomers thereof, where the —O—R3 groups may be independently located at any position on the terminal phenyl groups, where each R1 is independently selected from the group consisting of nothing, H, OH, C1-C12 (preferably C1-C4) alkyl groups, and halogens (such as I and Br), each R2 is independently selected form the group consisting of H, OH, and C1-C12 (preferably C1-C4) alkyl groups, and halogens (such as I and Br), each R3 group is independently selected from the group consisting of C1-C12 (preferably C1-C4) alkyl groups, and substituted or unsubstituted phenyl groups, and wherein the designation

refers to the fact that there may optionally be: 1) one or two non-fused double bonds at one or two valence-permitted positions around either or both of the six-membered, N-containing rings, such as illustrations a-d below; 2) a double bond between either or both of the N-containing rings and the adjacent carbons of the central moiety, with or without an additional non-fused double bond at any valence-permitted position around the corresponding N-containing ring, such as illustrations e-g. In instances of 2) where there is a double bond between the nitrogen atom of either N-containing ring and an adjacent carbon atom thereof, R1 is nothing, such as illustrations a-c and g. However, if there is no such nitrogen double bond, the corresponding R1 is selected from the group consisting of H, OH, and C1-C12 (preferably C1-C4) alkyl groups, such as illustrations d-f; or 3) either or both of the B rings are free of non-fused double bonds, and each R1 is independently selected from the group consisting of H, OH, and C1-C12 (preferably C1-C4) alkyl groups.

Set forth below are illustrations depicting certain exemplary double bond configurations of either or both of the N-containing rings of Structure V.

In other embodiments, the following compounds are useful

where R7 and R8 are attached at any position around the benzene ring and are independently selected from the group consisting of H, OH, and C1-C12 (preferably C1-C4) alkoxy groups, and where, preferably, R7 is OH, R8 is a C1-C12 (preferably C1-C4) alkoxy group.

Cinnamaldehyde/Harmaline 561 Compounds

Cinnamaldehyde occurs in the bark of cinnamon trees and is present in cis and trans isomers. It is identified by CAS #104-55-2.

These compounds are produced using the aldehyde reaction in the same fashion as the benzaldehyde/harmaline products using the aldehyde reactions, and have molecular weights of approximately 346, 328, and 542, as represented below. The MW 542 compound includes a first cinnamaldehyde moiety with two harmaline moieties bonded to the first moiety. The MW 346 compound is made up of a single cinnamaldehyde moiety and a single harmaline moiety, whereas the MW 328 product is a dehydrated version of the MW 346 product. The primary compound is the MW542 product.

Again, an analog of the above primary structure is a hydrogenated version wherein the N atoms of the two harmaline moieties are hydrogenated, eliminating the double bonds therein.

More broadly, however, appropriate cinnamaldehyde/harmaline compounds include one or more compounds of the structure:

and the dimers, isomers, and tautomers thereof, where the —O—R3 groups may be independently located at any position on the terminal phenyl groups, where each R1 is independently selected from the group consisting of nothing, H, OH, and C1-C12 (preferably C1-C4) alkyl groups, each R2 is independently selected form the group consisting of H, OH, and C1-C12 (preferably C1-C4) alkyl groups, each R3 group is independently selected from the group consisting of C1-C12 (preferably C1-C4) alkyl groups, and substituted or unsubstituted phenyl groups, and wherein the designation

refers to the fact that there may optionally be: 1) one or two non-fused double bonds at one or two valence-permitted positions around either or both of the six-membered, N-containing rings, such as illustrations a-d below; 2) a double bond between either or both of the N-containing rings and the adjacent carbons of the central moiety, with or without an additional non-fused double bond at any valence-permitted position around the corresponding N-containing ring, such as illustrations e-g. In instances of 2) where there is a double bond between the nitrogen atom of either N-containing ring and an adjacent carbon atom thereof, R1 is nothing, such as illustrations a-c and g. However, if there is no such nitrogen double bond, the corresponding R1 is selected from the group consisting of H, OH, and C1-C12 (preferably C1-C4) alkyl groups, such as illustrations d-f; or 3) either or both of the N-containing rings are free of non-fused double bonds and each R1 is independently selected from the group consisting of H, OH, and C1-C12 (preferably C1-C4) alkyl groups.

Set forth below are illustrations depicting certain exemplary double bond configurations of either or both of the N-containing rings of Structure VI.

Vanillin/Harmaline 562 Compounds

The aldehyde reactions between vanillin and harmaline components, carried out in the same fashion as the benzaldehyde/harmaline reaction, yield products as set forth below.

As explained in Example 14, a confirmed vanillin/harmaline compound is

An analog of the above structure involves hydrogenation of the N-containing ring nitrogen atoms and is set forth below:

More broadly, however, appropriate vanillin/harmaline compounds include one or more compounds of the structure:

and the dimers, isomers, and tautomers thereof, where each R4 is independently selected from the group consisting of nothing, H, OH, and C1-C12 (preferably C1-C4) alkyl groups, each R5 is independently selected from the group consisting of H, OH, and C1-C12 (preferably C1-C4) alkyl groups, each R6 group is independently located at any position around the corresponding terminal phenyl group, or at either of the two open positions of the two N-containing rings, and is selected from the group consisting of C1-C12 (preferably C1-C4) alkoxy groups, H, OH, and substituted or unsubstituted phenyl groups, R7 and R8 are attached at any position around the benzene ring and are independently selected from the group consisting of H, OH, and C1-C12 (preferably C1-C4) alkoxy groups, with the proviso that R7 and R8 are not both H, and where, preferably, R7 is OH, R8 is a C1-C12 (preferably C1-C4) alkoxy group, and each R9 is independently selected from the group consisting of H, OH, and C1-C12 (preferably C1-C4) alkyl groups, and wherein the designation

refers to the fact that there may optionally be: 1) zero, one, or two non-fused double bonds at one or two valence-permitted positions around either or both of the six-membered, N-containing rings, such as illustrations a′-d′ below; 2) a double bond between either or both of the N-containing rings and the adjacent carbons of the central moiety, with or without an additional non-fused double bond at any valence-permitted position around the corresponding N-containing ring, such as illustrations e′-g′ below. In instances of 2) where there is a double bond between the nitrogen atom of either N-containing ring and an adjacent carbon atom thereof, R4 is nothing, such as illustrations a′-c′ and g′ below. However, if there is no such nitrogen double bond, the corresponding R4 is selected from the group consisting of H, OH, and C1-C12 (preferably C1-C4) alkyl groups, such as illustrations d′-f; or 3) either or both of the N-containing rings are free of non-fused double bonds and each R4 is independently selected from the group consisting of H, OH, and C1-C12 (preferably C1-C4) alkyl groups.

Set forth below are illustrations depicting certain exemplary double bond configurations of either or both of the N-containing rings of Structure VII.

An exemplary compound consistent with 3) above is a hydrogenated form of the preferred 562 compound having the structure

Phenoxy Benzaldehyde/Harmaline 594 Compounds

As described in Example 24, the principal 594 compound has the following structure:

Orthovanillin/Harmaline 523 Compounds

Aldehyde reactions between orthovanillin and harmaline are quite diverse, and the resulting products are similarly variable. Four reaction schemes have been identified as potential candidates, as set forth below.

It will be observed that the foregoing scheme 4 compounds involve coupling between harmaline and orthovanillin via a pyrrole nitrogen linkage, i.e., the orthovanillin moieties bond to the nitrogen atom of the pyrrole ring forming a part of harmaline.

The initial aldehyde reaction between orthovanillin and harmaline of scheme 1 may also produce the following compound having the chemical formula C21H2ON2O3 and a molecular weight of 348.15. It will be observed that in this instance the initial reaction between orthovanillin and harmaline occurs at the cyclohexyl diene nitrogen atom.

The structure of a preferred orthovanillin/diharmaline compound is set forth below:

More broadly, however, preferred orthovanillin/diharmaline compounds are defined by Structure VIII below:

and the dimers, isomers, and tautomers thereof, where the —O—R3 groups may be independently located at any position on the terminal phenyl groups, where each R1 is independently selected from the group consisting of nothing, H, OH, and C1-C12 (preferably C1-C4) alkyl groups, each R2 is independently selected form the group consisting of H, OH, and C1-C12 (preferably C1-C4) alkyl groups, each R3 group is independently selected from the group consisting of C1-C12 (preferably C1-C4) alkyl groups, and substituted or unsubstituted phenyl groups, and wherein the designation

refers to the fact that there may optionally be: 1) one or two non-fused double bonds at one or two valence-permitted positions around either or both of the six-membered, N-containing rings, such as illustrations a-d below; 2) a double bond between either or both of the N-containing rings and the adjacent carbons of the central moiety, with or without an additional non-fused double bond at any valence-permitted position around the corresponding N-containing ring, such as illustrations e-g. In instances of 2) where there is a double bond between the nitrogen atom of either N-containing ring and an adjacent carbon atom thereof, R1 is nothing, such as illustrations a-c and g. However, if there is no such nitrogen double bond, the corresponding R1 is selected from the group consisting of H, OH, and C1-C12 (preferably C1-C4) alkyl groups, such as illustrations d-f; or 3) either or both of the N-containing rings are free of non-fused double bonds and each R1 is independently selected from the group consisting of H, OH, and C1-C12 (preferably C1-C4) alkyl groups.

Set forth below are illustrations depicting certain exemplary double bond configurations of either or both of the N-containing rings of structure VI.

One particular aldehyde reaction for preparing orthovanillin-harmaline compounds is to mix together solid particulate orthovanillin and harmaline at a weight ratio of orthovanillin:harmaline of about 2:1, followed by adding ethanol, DMSO, or a 90% ethanol/10% DMSO mixture to the particulates. Thereupon, the dispersion is agitated and allowed to stand for 24 hours at room temperature. The specific steps are: (1) mix together 500 mg of orthovanillin and 250 mg of harmaline in a 15 mL jar; (2) gently shake the jar until a uniform powder mixture is present; (3) add 10 mL of ethanol and/or DMSO to the dry mixture; (4) agitate with a vortex mixer at 1000 rpm for 10 minutes; and (5) let the dispersion set and the reaction proceed for 24 hours at room temperature.

A similar technique involving reactions between harmaline and vanillin comprises mixing particulate harmaline and vanillin together at a weight ratio of about 2:1 (vanillin:harmaline), followed by adding ethanol to a final concentration of the reactions of from about 10-100 mg/mL. This mixture is then allowed to sit for approximately 3 days at 50° C. A bluish solid forms, which is filtered and washed with methanol, and recovered.

Phenoxy Benzaldehyde/Harmaline 594 Compounds

The aldehyde reactions between phenoxy benzaldehyde and harmaline components, carried out in the same fashion as the benzaldehyde/harmaline reaction, yield products as set forth below.

and the dimers, isomers, and tautomers thereof, where the —O—R3 groups may be independently located at any position on the terminal phenyl groups, where each R1 is independently selected from the group consisting of nothing, H, OH, and C1-C12 (preferably C1-C4) alkyl groups, each R2 is independently selected form the group consisting of H, OH, and C1-C12 (preferably C1-C4) alkyl groups, each R3 group is independently selected from the group consisting of C1-C12 (preferably C1-C4) alkyl groups, and substituted or unsubstituted phenyl groups, and the phenoxy group may be substituted at any position on the benzyl ring, and wherein the designation

refers to the fact that there may optionally be: 1) one or two non-fused double bonds at one or two valence-permitted positions around either or both of the six-membered, N-containing rings, such as illustrations a-d below; or 2) a double bond between either or both of the N-containing rings and the adjacent carbons of the central moiety, with or without an additional non-fused double bond at any valence-permitted position around the corresponding N-containing ring, such as illustrations e-g. In instances of 2) where there is a double bond between the nitrogen atom of either N-containing ring and an adjacent carbon atom thereof, R1 is nothing, such as illustrations a-c and g. However, if there is no such nitrogen double bond, the corresponding R1 is selected from the group consisting of H, OH, and C1-C12 (preferably C1-C4) alkyl groups, such as illustrations d-f; or 3) either or both of the N-containing rings are free of non-fused double bonds and each R1 is independently selected from the group consisting of H, OH, and C1-C12 (preferably C1-C4) alkyl groups.

Set forth below are illustrations depicting certain exemplary double bond configurations of either or both of the N-containing rings of structure VII.

A particularly preferred compound of this type is set forth below and referenced in Example 24, where 3-phenoxybenzaldehyde was used.

Harmaline Components

Some harmaline components are tricyclic compounds of the structure

where the numbered 6-member fused ring is a N-heterocycle with a single N atom at any of the positions 2-5, and the R6 substituents may be located at any ring position; R5′ is H or C1-C12 (preferably C1-C4) alkoxy; and R6′ is H, a C1-C12 (preferably C1-C4) alkyl, or a C1-C12 (preferably C1-C4) carboxylic acid.

Representative compounds of this type include harmaline and the following:

In the foregoing representative compounds, any methoxy substituent may be replaced by a C2-C4 alkoxy group, or by a phenoxy group.

A number of other aldehydes have been reacted with harmaline to produce compounds, apart from those described above and detailed in the following examples. In each instance, the reaction was carried out by mixing together 500 mg of aldehyde and 250 mg of harmaline in a 15 mL jar, followed by gentle shaking the jar until a uniform powder mixture is present. Thereupon, 10 mL of ethanol was added to the dry mixture, the jar was capped, and was placed in a warm water bath of about 40° C. for approximately 24 hours.

In the table below, the specific harmaline-reacted aldehydes are identified together with the compounds obtained. As to the latter, the makeup of the respective compounds is identified by reactant moieties therein less any dehydration and/or reduction by virtue of the reaction, along with the approximate molecular weights thereof. For example, a given compound recited as “H+A−H2O” refers to a product containing one moiety of harmaline and one moiety of Aldehyde, minus one water molecule, whereas 2H+A−H20-2H refers to a product containing two moieties of harmaline, one moiety of aldehyde, less one water molecule and less two hydrogen atoms.

Compound Compound Aldehyde Makeup Molecular Weight o-anisaldehyde H + A − H2O 332 o-anisaldehyde 2H + A − H2O 546 o-anisaldehyde 2H + 2A − 2H2O 664 ethyl vanillin H + A − H2O 362 ethyl vanillin 2H + A − H2O 576 ethyl vanillin 2H + 2A − 2H2O 724 vanillin isobutyrate H + A − H2O 418 vanillin isobutyrate 2H + A − H2O 632 veratraldehyde H + A − H2O 362 veratraldehyde 2H + A − H2O 576 5-nitrovanillin H + A − H2O 393 vanillin acetate H + A − H2O 390 vanillin acetate 2H + A − H2O 604 vanillin acetate 2H + 2A − 2H2O 780 3-hydroxy-5-methoxybenzaldehyde H + A − H2O 348 3-hydroxy-5-methoxybenzaldehyde 2H + A − H2O 562 3-hydroxy-5-methoxybenzaldehyde 2H + 2A − 2H2O 696 2-hydroxy-4-methoxybenzaldehyde H + A − H2O 348 3-chloro-4-hydroxy-5-methoxybenzaldehyde H + A − H2O 382 3-chloro-4-hydroxy-5-methoxybenzaldehyde 2H + A − H2O 597 4-benzyloxy-3-methoxybenzaldehyde H + A − H2O 438 4-benzyloxy-3-methoxybenzaldehyde 2H + A − H2O 652 3,4-dimethoxy-5-hydroxybenzaldehyde H + A − H2O 378 3,4-dimethoxy-5-hydroxybenzaldehyde 2H + A − H2O 592 3,4-dimethoxy-5-hydroxybenzaldehyde 2H + 2A − 2H2O 756 5-bromovanillin H + A − H2O 427 5-bromovanillin 2H + A − H2O 641 2-bromo-3-hydroxy-4-methoxybenzaldehyde H + A − H2O 427 2-bromo-3-hydroxy-4-methoxybenzaldehyde 2H + A − H2O 641 3-hydroxy-2-iodo-4-methoxybenzaldehyde H + A − H2O 474 3-hydroxy-2-iodo-4-methoxybenzaldehyde 2H + A − H2O 688 m-anisaldehyde H + A − H2O 332 m-anisaldehyde 2H + A − H2O 546 3-phenoxybenzaldehyde H + A − H2O 394 3-phenoxybenzaldehyde 2H + A − H2O 608 4-phenoxybenzaldehyde H + A − H2O 394 4-phenoxybenzaldehyde 2H + A − H2O 608 biphenyl-3-carboxaldehyde H + A − H2O 378 biphenyl-3-carboxaldehyde 2H + A − H2O 592 4-fluoro-3-phenoxybenzaldehyde H + A − H2O 412 4-fluoro-3-phenoxybenzaldehyde 2H + A − H2O 626 3-fluorobenzaldehyde H + A − H2O 320 3-fluorobenzaldehyde 2H + A − H2O 534 3-fluorobenzaldehyde 2H + 2A − 2H2O 640 4-fluorobenzaldehyde H + A − H2O 320 4-fluorobenzaldehyde 2H + A − H2O 534 4-fluorobenzaldehyde 2H + 2A − 2H2O 640 3,5-fluorobenzaldehyde H + A − H2O 338 3,5-fluorobenzaldehyde H + A 356 3,5-fluorobenzaldehyde 2H + A − H2O 552 3,5-fluorobenzaldehyde 2H + 2A − 2H2O 676 2,4,5-fluorobenzaldehyde H + A − H2O 356 2,4,5-fluorobenzaldehyde 2H + A − H2O 570 2,3,4,5,6-fluorobenzaldehyde H + A − H2O 392 2,3,4,5,6-fluorobenzaldehyde 2H + A − H2O 606 2,3,4,5,6-fluorobenzaldehyde 2H + 2A − 2H2O 784 4-methylbenzaldehyde H + A − H2O 316 4-methylbenzaldehyde H + A 334 4-methylbenzaldehyde 2H + A − H2O 530 teraphthalaldehyde H + A − H2O 330 teraphthalaldehyde 2H + A − H2O 544 teraphthalaldehyde 2H + 2A − 2H2O 660 4-chlorobenzaldehyde H + A − H2O 336 4-chlorobenzaldehyde H + A 354 perillaldehyde H + A − H2O 346 perillaldehyde 2H + A − H2O 560 cuminaldehyde H + A − H2O 344 cuminaldehyde 2H + A − H2O 553 cuminaldehyde 2H + A 576 cuminaldehyde 2H + 2A − 2H20 688 cyclohexanecarboxaldehyde 2H + A − H2O − 2H 520 cyclohexanecarboxaldehyde H + A 326 cyclohexanecarboxaldehyde H + A − H20 308

The following are representative structures of some of the compounds set forth in the above table wherein two moieties of harmaline were reacted with the recited aldehydes (in some cases, the aldehydes are identified by different, equivalent names).

Additionally, number of harmaline-like compounds were reacted with different aldehydes to produce compounds, apart from those detailed in the following examples. In each instance, the reaction was carried out by mixing together 500 mg of a selected aldehyde and 250 mg of a harmaline-like compound in a 15 mL jar, followed by gentle shaking the jar until a uniform powder mixture is present. Thereupon, 10 mL of ethanol was added to the dry mixture, the jar was capped, and was placed in a warm water bath of about 40° C. for approximately 24 hours.

In the table below, the specific harmaline-like compounds and aldehydes are identified together with the compounds obtained. As to the latter, the makeup of the respective compounds is identified by reactant moieties therein less any dehydration by virtue of the reaction, along with the approximate molecular weights thereof. For example, a given compound recited as “H+A−H2O” refers to a product containing one moiety of harmaline-like compound and one moiety of aldehyde, minus one water molecule.

Harmaline-like Compound Compound Compound Aldehyde Makeup Molecular Weight 1 orthovanillin H + A − H2O 306 1 orthovanillin H + 2A − H2O 458 1 benzaldehyde H + 2A − H2O 366 2 orthovanillin H + A 366 2 orthovanillin H + A − H2O 348 2 orthovanillin 2H + A − H2O 562 2 orthovanillin 2H + 2A − 2H2O 696 3 orthovanillin H + A 352 4 orthovanillin H + A − H2O 336 4 orthovanillin H + 2A − H2O 488 5 orthovanillin H + A 354 5 orthovanillin H + A − H2O 336 5 orthovanillin 2H + A − H2O 538 6 benzaldehyde H + A − H2O 288 1 = 1,2,3,4-Tetrahydro-9H-pyrido[3,4-b]indole (THβC) 2 = 6-methoxy-1-methyl-3,4-dihydro-2H-yrido[3,4-b]indole (6-Methoxyharmalan) 3 = 4,9-dihydro-3H-beta-carbolin-1-yl methyl ether 4 = 6-Methoxy-1,2,3,4-tetrahydro-9H-pyrido[3,4-b]indole (pinoline) 5 = 2,3,4,5-tetrahydro-8-methoxy-lH-pyrido[4,3-b]indole 6 = 4,9-Dihydro-1-methyl-3H-pyrido[3,4-b]indol-7-ol hydrochloride (harmalol hydrochloride)

EXAMPLES

The following Examples set forth preferred therapeutic agents and methods in accordance with the invention, but it is to be understood that these examples are given by way of illustration only, and nothing therein should be taken as a limitation upon the overall scope of the invention.

Example 1

In this Example, a series of 523 compounds were prepared using the aldehyde reaction, comprising reacting respective quantities of solid synthetic orthovanillin (99% by weight purity) and synthetic harmaline (92% by weight purity). In each instance, the orthovanillin and harmaline reacted to give one or more compounds. These compositions are referred to as GZ523.001-008, and the makeup and formulation thereof are set forth below, with quantities and approximate percent-by-weight levels for these two components:

GZ523.001—294 mg orthovanillin (85.5%)+50 mg harmaline (14.5%), with 5 mL ethanol, mixed immediately;

GZ523.002—294 mg orthovanillin (85.5%)+50 mg harmaline (14.5%), with 5 mL DMSO, mixed immediately;

GZ523.003—229.3 mg orthovanillin (66.7%)+114.7 mg harmaline (33.3%) mixed together as dry ingredients and allowed to stand for 48 hours in a closed vessel, followed by the addition of 5 mL ethanol;

GZ523.004—286.7 mg orthovanillin (83.3%)+57.3 mg harmaline (16.7%) mixed together as dry ingredients and allowed to stand for 13 days, followed by the addition of 5 mL ethanol;

GZ523.005—229.3 mg orthovanillin (66.7%)+114.7 mg harmaline (33.3%), with 5 mL DMSO, mixed immediately, and allowed to stand for 24 hours;

GZ523.006—229.3 mg orthovanillin (66.7%)+114.7 mg harmaline (33.3%), with 5 mL ethanol, mixed immediately, and allowed to stand for 24 hours;

GZ523.007—172 mg orthovanillin (50%)+172 mg harmaline (50%), with 5 mL ethanol, mixed immediately, and allowed to stand for approximately 3 weeks; and

GZ523.008—229.3 mg orthovanillin (66.7%)+114.7 mg harmaline (33.3%) mixed together as dry ingredients, place in a closed vial for 45 minutes, followed by standing stand for 24 hours in a covered tray, followed by the addition of 5 mL ethanol.

Example 2

The 523 compounds of Example 1 were subjected to a series of identical in vitro assays against lymphoma (M0205) and leukemia (jurkat E6-1) cell lines, in order to determine the anti-cancer properties of the compositions, as determined by cell death. The protocol for the assays is given below.

Methods

The individual cells were grown in suspension in media (RPMI supplemented with 10% FBS), maintained at approximately 500,000 cells/mL. The cells were directly plated in 96-well plates, and each well was exposed to increasing doses of the GZ523.001-.008 compositions for 24 hours (a minimum of 4 replicates for each dosage). After a 24-hour exposure to the selected dosages of the test compositions, PrestoBlue (Life Technologies, Inc) was added to each well and fluorescence readings were taken 4-6 hours later with an excitation wavelength of 485 nm and an emission wavelength of 560 nm, using a microplate reader (Enspire Multimode, PerkinElmer). Results were averaged following background subtraction and normalized to untreated cell controls.

The results of these tests are set forth in FIGS. 1-16, where FIGS. 1-8 are the lymphoma test results and FIGS. 9-16 are the leukemia test results and, in each case, the compositions exhibited excellent anti-cancer activity at relatively low dosages. In general, dosages exceeding 10 μg/mL gave very good results, with extraordinary results above about 40 μg/mL.

Example 3

In this Example, a 523 compound was prepared containing orthovanillin and harmaline at a weight ratio of 2:1, using solid synthetic orthovanillin (99% pure) and solid synthetic harmaline (92% pure). The reactants were dispersed in ethanol to achieve a concentration 75 mg/mL, and allowed to react for a period of 24 hours. After the reaction was complete, the compound (designated as GZ523F001) was treated by HPLC to recover a high molecular weight fraction predominantly (about 70% by weight) made up of dioligomer(s) having a molecular weight of approximately 696, and unreacted harmaline. These dioligomer(s) included one or more compounds exemplified by the Scheme 1 dimers.

This compound was then tested against the same lymphoma and leukemia cells as set forth in Example 2. The results of this test are set forth in FIGS. 17 and 18. These results confirm that the compounds exhibited very significant anti-cancer activities.

Example 4

In this series of tests, non-Hodgkin lymphomas were tested for their susceptibility to a preferred compound in accordance with the invention, namely GZ523.006 described in Example 1. The cell lines were grown in suspension according to the vendors' instructions and tested by the methods described in Example 2, except that there were no replications. The following Table sets forth each subtype of non-Hodgkin lymphoma tested, the cell line ID number, and the median effective dose (EC₅₀). The EC₅₀ represents the potency of the GZ523.006 composition against the cell lines, and ranged from 8-38 μg/mL, which is considered a therapeutically appropriate dosage range. The effect size of the highest concentration of GZ523.006 determines how well the composition worked to directly kill the respective cells. For all of the cell lines tested, 100% of the cancer cells were dead at a dosage of 25 μg/mL or greater.

TABLE 1 Non-Hodgkin Lymphoma classification Cell ID EC₅₀ (μg/mL) Human, B cell (mantle cell) MO2058 26.12 Human, B cell DHL4 15.65 Human, B cell (diffuse large cell mixed, DHL6 13.36 and follicular) Human, B cell (diffuse large cell) DHL8 8.064 Human, B cell (diffuse large cell) DHL9 10.38 Human, B cell (diffuse histiocytic) DHL10 11.11 Human, B cell (diffuse large cell) DHL16 38.18 Human, B lymphoblast RL 17.82 Human, B cell HBL1 18.95 Human, lymphoblast (mantle cell) Mino 8.01 Human, B cell (mantle cell) Jvm13 14.88

Example 5

In this Example, a 562 compound was prepared by mixing 500 mg of vanillin powder and 250 mg of harmaline powder in a 15 mL jar. The powders were gently shaken to create a substantially uniform mixture, and 10 mL of dimethyl sulfoxide was added. The mixture was then agitated with a vortex mixer at 1000 rpm for 10 minutes to create a dispersion. In the case of one composition (GZ518.000), the dispersion compound(s) was tested immediately against lymphoma (M0205) by application to the cells, as described in Example 2. A second composition (GZ518.001) was prepared from the dispersion by allowing it to react for 24 hours at room temperature before testing against the lymphoma cells by application thereto. As set forth in FIGS. 19 and 20, both compositions and exhibited good anti-cancer properties.

Example 6

In this Example, the EC₅₀ values of GZ523.006 was determined for 25 different lymphoma cell lines. The experiments were performed using two-fold serial dilutions of GZ523.006 between 0.4 μg/mL and 100 μg/mL. Test wells were prepared using media and GZ523.006 controls for background subtraction. Each cell line was seeded with 10,000 cells/well, with triplicate technical replicates. After a 96-hour exposure, Alamar Blue Reagent (Life Technologies) was added to each well and incubated one hour at 37° C. Fluorescence values were recorded using a 560 nm excitation/590 nm emission filter set, and EC₅₀ concentrations were calculated using GraphPad Prism software. The EC₅₀ data for the 25 cell lines tested are set forth in FIG. 21, where: GCB-DLBCL are Germinal Center B-Cell-Diffuse Large B-Cell Lymphoma cell lines; ABC-DLBCL are Activated B-Cell-Diffuse Large B-Cell Lymphoma cell lines; MCL are Mantle Cell Lymphoma cell lines; and FL are Follicular Lymphoma cell lines. The error bars represent standard error of the mean values.

Each of the cell lines of FIG. 21 were incubated with 5, 10, or 20 μg/mL for 72 hours, then incubated Hoechst 33342 dye (BD Pharmigen) for 60 minutes at 37° C. The cells were washed twice and fluorescence data was acquired using a LSRII 4-laser flow cytometer (BD Biosciences). Data was analyzed using Flojo v10 and Modfit v4.05 software to quantify the percentage of dead cells (sub-G1), senescent cells (G1 peak), and cycling cells (S-phase and G2) in each condition. The data from this series of tests is summarized in Table 2.

TABLE 2 Percent of Live Cells Cell Line GZS23 (μg/ml) SubG1 % G1 G2 S SUDHL4 0 2.15 49.09 13.41 37.51 SUDHL4 5 1.81 50.5  14.03 35.47 SUDHL4 10 15.3 78.1  8.56 13.32 SUDHL4 20 90.3 N/A N/A N/A SUDHL6 0 3.35 40.67 23.99 35.34 SUDHL6 5 6.22 72.13 14.77 13.11 SUDHL6 10 44.6 86.25 9.58  4.17 SUDHL6 20 92.3 N/A N/A N/A SUDHL8 0 2.28 32.71 8.81 58.48 SUDHL8 5 1.66 35.59 9.49 54.92 SUDHL8 10 6.48 32.41 18.01 49.58 SUDHL8 20 88.1 N/A N/A N/A SUDHL9 0 2.42 29.2  22.39 48.41 SUDHL9 5 9.52 28.84 35.61 35.56 SUDHL9 10 57.9 27.43 0 72.57 SUDHL9 20 79.3 42.45 0 57.55 SUDHL10 0 6.05 30.76 16.38 52.86 SUDHL10 5 4.23 30.03 10.54 59.43 SUDHL10 10 8.14 31.86 11.11 57.03 SUDHL10 20 91.5 30.9  12.23 56.87 KARPASS422 0 7.92 42.06 14.98 42.96 KARPASS422 5 34.7 47.02 16.22 36.76 KARPASS422 10 77.3 N/A N/A N/A KARPASS422 20 96.2 N/A N/A N/A BJAB 0 3.79 27.53 27.45 45.01 BJAB 5 4.94 32.33 23.68 44   BJAB 10 20 29.11 22.23 48.66 BJAB 20 35.2 54.49 13.32 32.2  OCI-Ly1 0 3.37 53.29 11.19 35.52 OCI-Ly1 5 6.04 52.93 14.28 32.79 OCI-Ly1 10 6.64 52.06 14.21 33.73 OCI-Ly1 20 32.7 37.08 14.73 48.19 OCI-Ly7 0 10.4 29.77 17.25 52.99 OCI-Ly7 5 7.85 31.08 12.21 56.72 OCI-Ly7 10 12.7 33.42 11.94 54.64 OCI-Ly7 20 86.1 N/A N/A N/A OCI-Ly3 0 7.94 43.31 15.37 41.32 OCI-Ly3 5 15.9 62.11 7.34 30.54 OCI-Ly3 10 12.8 59.85 9.14 31.01 OCI-Ly3 20 60 53.31 10.82 35.87 U2932 0 2.01 59.86 19.61 20.53 U2932 5 4.61 66.42 16.48 17.09 U2932 10 9.61 69.8  16.14 14.06 U2932 20 28.5 51.28 23.97 24.76 HBL1 0 10.7 36.34 19.16 44.5  HBL1 5 16.3 70.39 14.87 14.73 HBL1 10 25.4 73.47 9.55 16.98 HBL1 20 85.1 N/A N/A N/A RL 0 3.49 31.15 25.23 43.62 RL 5 23.4 40.26 16.07 43.67 RL 10 30.2 44.22 15.17 40.61 RL 20 30.2 50.11 23.31 26.57 DoHH2 0 2.71 37.26 15.73 47.01 DoHH2 5 9.45 71.44 9.12 19.44 DoHH2 10 67.6 55.75 11.86 32.39 DoHH2 20 84.1 41.51 19.14 39.34 FL18 0 5.5 34.8  16.47 48.73 FL18 5 3.13 30.55 20.78 48.67 FL18 10 14.1 34.08 20.46 45.45 FL18 20 90.4 N/A N/A N/A WSU-FSCCL 0 2.12 42.98 14.12 42.89 WSU-FSCCL 5 3.38 45.67 15.31 39.02 WSU-FSCCL 10 N/A N/A N/A N/A WSU-FSCCL 20 49.7 N/A N/A N/A JVM13 0 5.2 53.26 10.27 36.47 JVM13 5 6.18 56.27 12.25 31.48 JVM13 10 7.99 59.61 11.5 28.88 JVM13 20 47.5 71.02 6.26 22.72 JEKO 0 10.6 54.04 4.86 41.1  JEKO 5 9.89 46.76 7.02 46.22 JEKO 10 73.3 21.36 14.55 64.09 JEKO 20 96.9 N/A N/A N/A L128 0 2.97 43.38 4.74 51.88 L128 5 4.26 59.46 6.3 34.24 L128 10 8.35 70.13 6.99 22.87 L128 20 72.7 N/A N/A N/A MINO 0 2.2 44.64 5.38 49.98 MINO 5 2.79 59.12 7.81 33.07 MINO 10 3.74 65.18 7.96 26.86 MINO 20 70 64.83 6 29.18 NCEBI 0 2.99 41.89 16.51 41.59 NCEBI 5 6.52 41.1  15.94 42.96 NCEBI 10 12.8 48.57 11.19 40.24 NCEBI 20 45.8 32.41 10.59 57   GRANTA519 0 8.23 41.15 14.66 44.18 GRANTA519 5 10.1 41.57 14.49 43.93 GRANTA519 10 10.7 41.57 11.54 46.89 GRANTA519 20 33.3 N/A N/A N/A JVM2 0 9.04 55.42 24.5 20.08 JVM2 5 6.68 57.93 26.86 15.21 JVM2 10 9.05 59.39 26.53 14.07 JVM2 20 36.9 65.19 18.18 16.63

The mechanism of GZ523.0006 cell death was interrogated using Annexin V and 7-AAD staining, with the BD Apoptosis Detection Kit (BD Pharmigen). Four cell lines were selected that showed high sensitivity to GZ523.006. The cell lines were incubated with 5, 10, or 20 μg/mL of GZ523.006 for 72 hours, washed, re-suspended in 1× Annexin V Binding Buffer, and stained with PE-Annexin V and 7-AAD for 15 minutes at room temperature in the dark. The cells were then suspended in additional binding buffer and analyzed using an LSRII 4-layer flow cytometer (BD Biosciences). Data were analyzed using Flojo v10, by gating on untreated cells. The summary of results of this experiment are set forth on Table 3.

TABLE 3 GZ523 Annexin-V negative Annexin-V positive Annexin-V positive Annexin-V negative Cell line (μg/ml} 7AAD positive 7 AAD positive 7AAD negative 7 AAD negative DoHH2 0 0.74 1.96 2.38 94.9 DoHH2 5 63.6 20.9 5.58 9.86 DoHH2 10 48.2 44.9 5.26 1.55 DoHH2 20 51 48.2 0.071 0.71 SUDHL4 0 0.77 0.47 1.99 96.8 SUDHL4 5 1.7 0.82 1.44 96 SUDHL4 10 11.7 2.09 2.08 84.2 SUDHL4 20 2.59 82.2 11.7 3.45 KARPAS422 0 0.73 2.31 4.13 92.8 KARPAS422 5 8.3 17.5 11.9 62.3 KARPAS422 10 14.5 12.9 7.92 64.7 KARPAS422 20 41.3 45.4 8.28 5.06 RL 0 1.49 0.68 2.24 95.6 RL 5 9.93 2.88 2.62 84.6 RL 10 21.4 3.56 1.7 73.3 RL 20 24.4 69.1 3.2 3.31

A Caspace 3/7 assay was performed by induction of apoptosis using a luminescence-based Caspace cleavage assay. Cells were plated at 10,000 cells per well and exposed to 20 μg/mL of GZ523.006 for 48 hours. Caspace activation was measured using the Caspace-Glo 3/7 assay (Promega), and compared with vehicle control exposures. Caspace activation was measured with triplicate technical replicates and duplicate experimental replicates using a luminescence plate reader. These test results are summarized in FIG. 22, where Bar 1 is the control and Bar 2 is treated cells. The error bars represent standard error of the mean values. These tests confirm that GZ523.006 exhibits cytotoxic properties against lymphoma cell lines to the induction of apoptosis. Mantle cell lymphoma cell lines exhibited the highest resistance, while Diffuse Large B-Cell Lymphomas of the Germinal Center B-Cell-like subtype exhibited the greatest sensitivity.

Example 7

1 Objective

The objective of this study was to determine the maximum tolerated dose and potential toxicity of GZ523.010 following 7 days of daily oral administration in CD1 mice. GZ523.010 was prepared by mixing 2433 mg orthovanillin, 1217 mg harmaline, and 5 mL of ethanol. This mixture was then sonicated at 35° C. for one hour to assure complete mixing, and was then allowed to stand for 24 hours at room temperature.

2 Study Overview

There were four dose groups consisting of a vehicle control group and 3 dose groups for 7 days of repeated oral gavage dosing with 10 mice/sex/group. Animals were dosed once daily with GZ523.010 and euthanized on study Day 8. All animals were observed daily for any clinical signs after dose administration. Gross necropsy was conducted for each animal and clinical pathology was performed for all available samples at termination. The first day of dosing was defined as study Day 1. The study design and variables evaluated are presented in Table 4 and Table 5.

TABLE 4 Study Design No. of Dose Level Dose Volume Dose Group Animals Test Article (mg/kg) (mL/kg) Route/Frequency 1 (Control) 10/sex Vehicle 0 20 Oral Gavage/Once 2 (Low) 10/sex GZ523.010 50 20 daily for 7 days 3 (Mid) 10/sex GZ523.010 100 20 4 (High) 10/sex GZ523.010 300 20

TABLE 5 Variables Evaluated and Intervals Parameters Intervals Mortality Observation Twice daily Physical Examination Once during acclimation Body Weight Daily Food Consumption Daily (group average) Clinical Observations Twice daily Clinical pathology Hematology, coagulation & serum chemistry for three available animals from each gender per group Gross necropsy All animals with full list of tissues reserved for future analysis

3 Materials and Methods

3.1 GZ523.010

Test/Control GZ523.010 Distilled Water Article Name: Lot/Batch No: 20160530.1500 S1277 Storage Condition: 4-8° C. Room temperature Manufacturer: NA (sponsor provided) Southern Beverage Packers Components: 730 mg/mL GZ523.010 water in ethanol

3.2 Test System

3.2.1 Animals, Housing, and Environmental Conditions

Species/Strain: Mus Musculus, CD-1 mice Source: Charles River Number and Gender: 40 male and 40 female Age: 7.6 weeks at dose initiation Weight Range (Day −1): 21.1-33.6 g at dose initiation Identification: Ear notch and cage card Acclimation: 8 days Caging: polycarbonate shoebox cages Number Per Cage: 2-3 Environmental Conditions: Temperature: 20-28° C. (68-82.4° F.) Photoperiod: 12 hours light/12 hours dark Medication: No additional medication Randomization: Randomized per body weight within gender

3.2.2 Diet and Water

Diet: Type Rodent pellet diet Name Harlan rodent diet certified, lot#: 012816MA Availability ad libitum Water: Sources Standard facility deep well Availability ad libitum via Lixit Analysis for No coliform bacteria, E. coli, or Contaminants heavy metals reported Comments No contaminants were detected in the water at levels that would be expected to interfere with study results.

3.3 Dose Procedure

All animals were dosed via oral gavage once daily for 7 days according to Table 4. The dose volume was calculated based on the most recent body weights. Food and water were provided during the entire study period.

3.4 Mortality/Moribundity

General in-cage observations for mortality/moribundity were made twice daily.

3.5 Physical Examinations

All study animals were given physical examinations by qualified personnel once during acclimation to determine study eligibility, and again prior to termination. Examinations included, but were not limited to, examination of the skin and external ears, eyes, abdomen, neurological, behavior, and general body condition.

3.6 Clinical Observations

Detailed clinical observations were performed twice daily. The animals were observed for any signs of illness or reaction to treatment. Records of appearance, change, or disappearance of clinical signs were maintained on clinical observation sheets for each individual observation time point.

3.7 Body Weights and Food Consumption

All study animals were weighed daily during Day −1 through termination on Day 8. Group average food consumption was recorded daily from Day −1 through Day 7.

3.8 Termination and Necropsy

All animals were euthanized with CO2 at termination. Necropsy was performed on each animal and all designated issues/organs were collected for future potential analysis. The following tissues (when present), except testes and eyes, were preserved in 10% neutral-buffered formalin. Testes were fixed in modified Davidson's solution and eyes in Davidson's solution. Collected tissues were preserved for further evaluation.

adrenal(2) ovary (2) aorta pancreas bone (femur & sternum with marrow) pituitary gland brain (cerebellum, cerebrum, pons) prostate cecum rectum colon salivary gland [mandibular (2)] duodenum sciatic nerve epididymis (2) seminal vesicles esophagus skeletal muscle (quadriceps femoris) eyes, (including optic nerve) skin on abdominal region heart spinal cord (cervical, thoracic, lumbar) ileum spleen jejunum Sternum with bone marrow kidney (2) stomach lesions* testes (2) liver thymus lung with main stem bronchi thyroid (2) (parathyroid) lymph nodes (mandibular) trachea lymph node (cover dose site) urinary bladder mammary gland (females) uterus (+cervix + oviducts) & vagina *Gross lesions were collected at the discretion of the personnel conducting the necropsy.

3.9 Clinical Pathology

Clinical pathology was performed at termination. The clinical pathology analysis was performed on all designated animals that were euthanized on schedule. Animals were food fast overnight.

Serum Chemistry: Blood samples when available (˜0.5 mL) were collected from three study animals per gender from each group, and allowed to clot for 15 minutes in room temperature. No anticoagulant was used. Serum samples were prepared by centrifuging at 3000 RPM for ˜15 minutes. The serum chemistry includes with priority (√). When a sample was insufficient for analysis, several samples from same group were pooled together:

Alanine aminotransferase (ALT) √ Creatinine √ Creatine Kinase √ Albumin √ Globulin Alkaline Phosphatase (ALP) √ Glucose Aspartate Aminotransferase (AST) √ Inorganic Phosphorus Gamma Glutamyltransferase (GGT) Potassium √ Blood Urea Nitrogen (BUN) √ Sodium √ Calcium Total Bilirubin Chloride Total Protein Cholesterol Triglycerides

Coagulation: Blood samples (˜0.4 mL/animal) when available were collected from three study animals per gender from each group. Sodium citrate (3.2%) was used as the anticoagulant. Plasma was prepared by centrifuging for approximately 15 minutes at 3000 rpm at 4° C. The blood coagulation analysis included, but was not limited to:

Activated Partial Thromboplastin Time (APTT) Prothrombin Time (PT)

Hematology Analysis: Blood samples when available (˜0.4 mL) were collected from three study animals per gender from each group. K3-EDTA was used as anticoagulant. The hematology analysis includes (with priority (I)):

White Blood Cell Count √ Differential White Blood Cell Count Red Blood Cell Count √ Neutrophils (% and absolute) RDW (Red Cell Distribution Eosinophils (% and absolute) Width) √ Hemoglobin√ Hematocrit√ Basophils (% and absolute) Mean Cell Volume √ Lymphocytes (% and absolute) √ Mean Cell Hemoglobin √ Monocytes (% and absolute) √ Mean Cell Hemoglobin Platelet Count √ Concentration √ MPV (mean platelet volume) √

4 Results

4.1 Dose Administration

The dose administrations are summarized in Table 6. All study animals were successfully administered with the target amount of vehicle or test article formulation. All dose formulations were prepared prior to dose administration. Before preparing the dose formulation, it was observed that the stock test article (730 mg/mL) was not a uniform consistency. Therefore, a dose concentration of 100 mg/mL was not able to be formulated due to large amount of precipitates. The protocol was amended to decrease the dose concentration. The stock formulation was warmed to room temperature and rigorously agitated with sonication to reach a uniform (mud like) consistency. This was then diluted to 73 mg/mL with ethanol (secondary stock). The secondary stock was used to prepare each final dose formulation. The final consistency of dose formulation appeared to be a suspension and was mixed thoroughly before dosing.

TABLE 6 Summary Dose Administration-Actual Dose Level Gender Male Female Group 1 2 3 4 1 2 3 4 Dose Level 0 50 100 300 0 50 100 300 (mg/kg/day) (Vehicle) (Low) (Mid) (High) (Vehicle) (Low) (Mid) (High) N 10 11 10 10 10/9^(a) 9^(b) 10 10 Day 1 0 50.0 99.5 291.6 0 50.0 100.8 298.9 Day 2 0 51.5 122.5 311.4 0 38.9 94.6 241.8 Day 3 0 49.9 99.6 300.4 0 50.4 101.0 293.2 Day 4 0 50.9 98.9 304.3 0 50.9 99.8 289.9 Day 5 0 50.2 101.6 296.9 0 50.6 100.0 298.1 Day 6 0 49.5 100.7 294.3 0 50.4 99.8 292.7 Day 7 0 49.4 100.4 293.2 0 51.3 98.9 298.9 Mean 0 50.2 103.3 298.9 0 48.9 99.3 287.6 ^(a)One mouse (1F17:12-0) was euthanized prior to dose on day 6 due to tail injury; ^(b)One mouse was identified to be male at termination (suspected to be misidentified at shipping).

4.2 Mortality/Moribundity

There were no observed instances of mortality or significant moribundity during the study period.

4.3 Physical Examinations

All study animals underwent physical examinations by a veterinarian once during acclimation and again prior to termination. All animals were generally healthy and deemed suitable for study inclusion.

4.4 Clinical Observations

Clinical observation findings are listed in Table 7. There were no test article-related findings during the exposure period following daily oral gavage dose administration.

TABLE 7 Group Summary Clinical Observation Findings Study Animal Study ID ID Day AM/PM Clinical Observations 1F17 12-0 6 AM & PM Animal found with slough tail, ~0.5 inch of tail was de-gloved; animal was euthanized 3M6 37-1 7 PM Small laceration, tail tip 4M10 40-1 4 PM Bottom right tooth broken 4M10 40-1 5 AM & PM Broken right bottom incisor

4.5 Body Weights

Group summary body weight and weight change results are presented in Tables 8 and 9. Over the course of the study, most study animals generally gained weight, especially for males. The weights of females retained or slightly decreased and there were no remarkable differences among groups.

TABLE 8 Group Summary Body Weight Results (g) Gender Male Female Group 1 2 3 4 1 2 3 4 Dose Level 0 50 100 300 0 50 100 300 (mg/kg/day) (Veh) (Low) (Mid) (High) (Veh) (Low) (Mid) (High) N 10 11 10 10 10/9 9 10 10 Day (−1) Mean 30.5 30.0 29.1 28.8 25.9 25.1 25.8 25.0 SD 1.8 1.9 2.1 2.2 1.0 1.2 1.5 2.3 Day 1 Mean 24.0 29.2 23.7 27.9 25.9 32.2 26.4 31.0 SD 0.6 1.2 1.2 0.9 0.7 0.9 0.7 1.3 Day 2 Mean 31.3 30.0 29.1 29.5 25.7 24.3 25.3 25.1 SD 2.0 2.3 2.2 2.3 1.4 1.2 1.7 2.4 Day 3 Mean 30.9 31.0 29.8 29.6 25.5 24.7 25.6 24.9 SD 2.1 2.5 2.0 2.6 1.3 1.2 1.5 2.2 Day 4 Mean 31.1 31.4 30.0 29.4 25.6 24.8 25.5 25.1 SD 2.0 2.3 2.3 2.5 1.1 1.4 1.6 2.4 Day 5 Mean 31.5 31.9 30.3 29.6 25.9 24.8 26.1 25.1 SD 2.0 2.6 2.3 2.7 1.1 1.2 1.8 2.4 Day 6 Mean 31.4 31.8 30.4 29.7 25.7 24.5 25.8 25.1 SD 2.3 2.6 2.2 2.7 1.4 1.3 1.5 2.2 Day 7 Mean 31.7 32.0 30.6 29.6 25.7 24.8 25.7 25.1 SD 2.1 2.4 2.1 2.9 1.4 1.4 1.3 2.2 Day 8^(b) Mean 28.5 28.4 27.3 27.2 23.4 22.1 23.3 23.1 SD 1.0 2.3 2.3 2.4 1.4 1.2 1.3 2.0 ^(a) One mouse (1F17:12-0) was euthanized prior to dose on day 6 due to tail injury. ^(b)Animals were fasted overnight.

TABLE 9 Group Summary Body Weight Change Results (g/day) Gender Male Female Group 1 2 3 4 1 2 3 4 Dose Level 0 50 100 300 0 50 100 300 (mg/kg/day) (Veh) (Low) (Mid) (High) (Veh) (Low) (Mid) (High) N 10 11 10 10 10/9 9a 10 10 Day (−1 to 1) Mean −6.56 −0.85 −5.32 −0.87 0.01 7.07 0.61 5.98 SD 2.01 2.35 2.56 2.25 1.36 1.48 1.59 2.58 Day 1 to 2 Mean 7.31 0.86 5.37 1.52 −0.20 −7.91 −1.16 −5.94 SD 2.21 2.69 2.45 2.42 1.42 1.68 1.80 2.68 Day 2 to 3 Mean −0.41 0.93 0.70 0.10 −0.23 0.44 0.31 −0.22 SD 0.60 0.72 0.80 0.58 0.54 0.67 0.44 0.64 Day 3 to 4 Mean 0.22 0.40 0.19 −0.20 0.15 0.10 −0.04 0.24 SD 0.41 0.57 0.41 0.45 0.62 0.45 0.55 0.58 Day 4 to 5 Mean 0.38 0.57 0.27 0.28 0.25 0.02 0.56 0.03 SD 0.33 0.49 0.27 0.34 0.60 0.44 0.56 0.48 Day 5 to 6 Mean −0.08 −0.10 0.11 0.04 −0.18 −0.37 −0.27 0.02 SD 0.38 0.39 0.27 0.41 0.55 1.01 0.88 0.72 Day 6 to 7 Mean 0.34 0.19 0.19 −0.07 0.07 0.31 −0.18 −0.07 SD 0.31 0.35 1.45 0.31 0.41 0.58 0.64 0.53 Day 7 to 8 Mean −3.22 −3.64 −3.23 −2.43 −2.33 −2.68 −2.36 −1.98 SD 0.34 0.49 1.29 0.47 0.43 0.38 0.45 0.46 Day (−1 to 8)^(b) Mean −0.25 −0.21 −0.22 −0.21 −0.31 −0.38 −0.32 −0.24 SD 0.09 0.12 0.11 0.11 0.13 0.15 0.10 0.09 aOne mouse (1F17:12-0) was euthanized prior to dose on day 6 due to tail injury. ^(b)Animals were fasted overnight.

4.6 Food Consumption

Group food consumption summary results are presented in Table 10. Over the course of the study, study animals generally had similar food consumption. Overall, there were no remarkable differences among groups.

TABLE 10 Group Summary Food Consumption (g/day) Gender Male Female Group 1 2 3 4 1 2 3 4 Dose Level 0 50 100 300 0 50 100 300 (mg/kg/day) (Veh) (Low) (Mid) (High) (Veh) (Low) (Mid) (High) N 10 11 10 10 10/9 9 10 10 Day 1 Mean 5.5 5.2 9.2 10.6 4.0 4.1 6.6 9.3 SD 0.5 0.6 1.6 1.5 0.5 0.6 2.7 1.1 Day 2 Mean 4.5 5.1 4.6 5.6 4.0 3.6 3.7 4.3 SD 0.3 0.9 1.0 2.3 0.7 0.8 0.7 0.7 Day 3 Mean 5.0 4.4 4.3 12.7 3.2 3.4 3.5 3.1 SD 0.5 0.6 0.5 14.4 0.6 0.8 0.9 1.0 Day 4 Mean 4.4 5.3 4.8 4.4 4.4 4.0 4.5 4.0 SD 0.5 0.8 0.6 0.8 0.8 1.1 0.4 0.5 Day 5 Mean 6.4 5.0 4.5 4.6 4.1 3.6 8.4 3.7 SD 4.2 0.5 0.1 1.7 1.3 0.5 8.7 0.6 Day 6 Mean 4.0 5.1 4.7 4.5 3.6 3.5 7.3 3.8 SD 2.2 0.3 0.5 1.3 0.7 0.9 7.2 0.8 Day 7 Mean 4.7 5.8 4.4 3.5 6.6 3.8 7.5 7.3 SD 1.0 3.3 0.2 0.4 2.8 0.5 5.2 3.3 Day 8^(a) Mean 1.1 1.0 0.7 0.6 1.8 0.4 5.4 2.1 SD 0.9 1.6 0.5 0.1 4.3 0.2 10.3 0.9 ^(a)Food consumed before fasting.

4.7 Clinical Pathology

Blood samples were collected from euthanized mice for hematology and serum chemistry analysis. Some blood samples (serum) did not have sufficient volume to complete all target parameter analyses.

Hematology and coagulation data are summarized in Table 11. When compared to the control group (Group 1), none of the hematology or coagulation parameters appeared to be affected in the mice that were treated with test article at different dose levels.

Serum chemistry data are summarized in Table 12. All serum chemistry results appeared within normal ranges. When compared to the control group (Group 1), none of the serum chemistry parameters appeared to be affected in the mice that were treated with test article formulation at different dose levels.

TABLE 11 Group Mean Hematology and Coagulation at Termination Male Female Gender 1 2 3 4 1 2 3 4 Group 0 50 100 300 0 50 100 300 Dose Level (mg/kg/day) (Veh) (Low) (Mid) (High) (Veh) (Low) (Mid) (High) N 4 3 3 3 3 3 3 3 Red Blood Cells RBC 10⁶/μL 10.15 9.80 10.68 10.56 10.37 10.53 9.76 10.07 HCT % 60.6 57.9 61.7 60.4 60.8 59.7 57.5 60.1 HGB g/dL 17.1 16.5 17.2 17.2 17.7 18.4 16.9 17.5 MCV fL 59.8 59.2 57.8 57.3 58.6 56.6 58.9 59.8 MCH pg 16.8 16.8 16.1 16.3 17.1 17.6 17.3 17.4 MCHC g/dL 28.2 28.5 27.9 28.5 29.2 31.2 29.4 29.2 White Blood Cells WBC 10/μL 7.49 6.07 8.67 6.00 7.23 10.03 6.73 6.01 Neutrophils 10/μL 1.06 0.93 1.62 0.82 1.46 1.25 0.74 0.86 % 14.3 15.8 18.4 13.4 20.2 11.8 10.8 14.4 Lymphocytes 10/μL 4.98 3.84 5.75 3.83 4.13 7.03 4.70 3.48 % 68.4 61.9 66.8 62.2 57.3 71.8 69.6 56.3 Monocytes 10/μL 0.09 0.09 0.08 0.06 0.07 0.08 0.06 0.04 % 1.2 1.5 0.9 1.0 0.9 0.7 0.9 0.8 Eosinophils 10/μL 1.32 1.10 0.94 1.23 1.44 1.51 0.99 1.42 % 15.3 19.1 10.8 20.6 19.8 13.8 15.5 25.3 Basophils 10/μL 0.03 0.01 0.05 0.02 0.04 0.07 0.03 0.03 % 0.4 0.2 0.6 0.3 0.5 0.8 0.5 0.6 LUC 10/μL 0.07 0.10 0.22 0.04 0.10 0.09 0.20 0.16 % 0.9 1.5 2.6 0.7 1.3 1.0 2.8 2.6 Clotting Potential Platelets 10/μL 1323 1103 1365 1357 658 1197 784 884 Coagulation N 3 5 4 3 3 2 3 3 PT sec 11.3 14.1 11.0 10.0 10.7 11.7 10.5 10.8 APTT sec 29.6 33.2 35.6 27.4 29.9 39.4 28.8 27.6

TABLE 12 Group Mean Serum Chemistry at Termination Gender Male Female Group 1 2 3 4 1 2 3 4 Dose Level (mg/kg/day) 0 50 100 300 0 50 100 300 N (Veh) (Low) (Mid) (High) (Veh) (Low) (Mid) (High) Electrolyte Balance 6 4 4 6 6 4 4 6 Sodium mEq/L 153 152 155 155 152 153 155 156 Potassium mEq/L 7.7 9.3 9.7 9.5 9.0 8.6 9.8 9.1 Chloride mEq/L 106 106 111 111 109 104 110 110 Calcium mg/dL 10.6 10.7 10.7 10.9 10.5 11.0 11.0 10.9 Phosphorus mg/dL . 9.9 11.5 10.4 9.9 10.1 11.1 . Carbohydrate Metabolism Glucose mg/dL 133 112 145 136 92 125 102 113 Liver Function a) Hepatocellular ALT U/L 23 25 39 42 64 40 30 33 AST U/L 100 97 269 218 223 199 119 203 Liver Function b) Hepatobiliary ALP U/L 134 150 124 139 111 153 119 156 T. Bilirubin mg/dL 0.2 0.5 0.2 0.3 0.3 0.3 0.2 0.2 Kidney Function Creatinine mg/dL 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 BUN mg/dL 21 19 26 25 19 . 18 16 Others T. Protein g/dL 5.0 5.7 5.5 5.6 5.8 5.8 5.7 5.6 Albumin g/dL 3.3 3.4 3.2 3.4 3.5 3.5 3.5 3.5 Globulin g/dL 1.7 2.4 2.2 2.3 2.3 2.3 2.2 2.2 A/G ratio 2.4 1.5 1.5 1.5 1.6 1.5 1.7 1.6 Cholesterol mg/dL 212 277 174 219 126 . 122 . Triglycerides mg/dL 94 155 95 163 135 106 73 130 GGT U/L 3 5 3 3 3 3 3 3 “.” indicates an insufficient sample volume

4.8 Necropsy and Tissue Collection

A complete necropsy was conducted on all study animals. Necropsy included examination of the external surface, all orifices, and cranial, thoracic, abdominal, and pelvic cavities including contents. Macroscopic findings are summarized in Table 13. All findings were considered to be incidental, and unrelated to the test article administration. All tissues were collected, including the entire remaining carcasses, and fixed for future potential evaluation.

TABLE 13 Individual Animal Necropsy Findings Study Animal Study ID ID Day Findings 3F12  3-1 8 ~3 mm cyst left ovary filled with serosanguineous fluid 3F17 15-0 8 Right ovary very small, no follicle seen 4F15 14-0 8 Both ovaries surrounded by thin walled cyst containing clear fluid

5 Summary and Conclusions

The study was conducted to determine the maximum tolerated dose and potential toxicity of the test article following 7 days of daily oral administration in CD1 mice. There were four dose groups consisting of a vehicle control group and 3 dose groups. Treatments were administered for 7 days as repeated oral gavage doses, with 10 mice/sex/group. All animals were successfully dosed once daily as proposed and euthanized on study Day 8. All animals were observed daily for any clinical signs following dose administration. Gross necropsy was conducted for each animal and clinical pathology was performed for all available samples at the termination.

There were no unscheduled deaths and no significant observations of moribundity during the study period. In general, all animals had normal food consumption and gained weight as expected over the course of the study. There were no test article-related clinical findings. Clinical pathology analysis and necropsy at termination showed that all study animals were in normal conditions.

In conclusion, animals tolerated the doses of GZ523.010 up to 300 mg/kg/day via oral administration daily for 7 days. Under the conditions of this study, the No-Observed Adverse Effect Level (NOAEL) was determined to be 300 mg/kg/day.

Example 8

In this Example, in vitro cell proliferation assays were performed using: (1) Human Myeloma tumor cell lines; (2) Human Lymphoma tumor cell lines; (3) Solid Human tumor cell lines; and (4) Parental, Lenalidomide resistant and Bortezomib resistant Jeko-1 Mantle cell Lymphoma tumor cell lines. The compounds tested were three diharmaline compounds, namely the 518B562 (or simply 562), 560, and 561 compounds. In addition, a monoharmaline product designated as 518F014 was also tested. This monoharmaline product had the following structure:

The 518B562 compound was prepared by mixing together particulate harmaline and vanillin at a weight ratio of about 2:1 (vanillin:harmaline), followed by adding ethanol to a final concentration of the reactions of from about 10-100 mg/mL. This mixture is then allowed to sit for approximately 3 days at 50° C. A bluish solid forms, which is filtered and washed with methanol, and recovered. It was found that addition of an acid such as hydrochloric acid to reduce the pH of the product, increased the solubility thereof.

The 560 compound was prepared by mixing together 500 mg of benzaldehyde and 250 mg of harmaline in a 15 mL jar followed by shaking. 10 mL of DSMO was then added to the mixture, followed by agitation using a vortex mixer at 1000 rpm for 10 minutes. The vortexed mixture was then allowed to set for 24 hours at room temperature. The resultant product was an orange liquid containing 10.6 mM compound, and was stored at 4° C. until use.

The 561 compound was prepared by mixing together 500 mg of cinnamaldehyde and 250 mg of harmaline in a 15 mL jar followed by shaking. 10 mL of DSMO was then added to the mixture, followed by agitation using a vortex mixer at 1000 rpm for 10 minutes. The vortexed mixture was then allowed to set for 24 hours at room temperature. The resultant product was an orange solid dispersed in liquid containing 10 mM compound, and was stored at 4° C. until use.

Each proliferation assay was carried out as follows. The test cells were plated in growth media using a 384-well microtiter plate at 50 μL volume. The cells were incubated for 24 hours at 37° C. in a humidified incubator. After 24 hours of incubation, the test compounds were added to the test wells in DSMO solvent, at concentrations ranging from 0.0075-100 μM. Control wells received equal volumes of DSMO, without compound. Following drugging, the cells were incubated for 72 hours at 37° C. in a humidified incubator. After this exposure, 100 μL of a 1:1 mixture of sterile water and CellTiter-Glo® reagent (Promega) was added to each well. The plates were then incubated for 60 minutes at room temperature, followed by recording the luminescence value of each well using a luminometer as a measure of cell proliferation.

The following Table 14 sets forth the cell lines tested using the respective compounds, a summary of the IC₅₀ Results, and an identification of the corresponding graphical Figure for each assay.

TABLE 14 In Vitro Cell Proliferation Assays Using 518B562, 560, and 561 Compounds with Summary of IC₅₀ Results Mean IC₅₀ Mean IC₅₀ Mean IC₅₀ Mean IC₅₀ (μM) (μM) Fig- (μM) (μM) Cell Line Tissue Type 518F014 518B562 No. 560 Fig. No. 561 Fig. No. MIA PaCa-2 Pancreatic >100     1.811 23 3.379 55 33.97 87 ASPC-1 87.13* 2.152 24 3.617 56 40.50 88 BxPC-3 98.65* 1.599 25 2.621 57 17.70 89 AN3CA Endometrial 88.10* 1.791 26 2.706 58 12.45 90 HEC-1a >100     4.752 27 5.097 59 75.96 91 MDA-MB-231 Triple 78.23* 1.625 28 3.891 60 48.47 92 MDA-MB-468 Negative >100     2.049 29 3.747 61 35.60 93 HCC70 Breast >100     3.454 30 5.943 62 54.37 94 H1975 (EGFR mut) Non Small 79.71* 1.840 31 4.272 63 58.29 95 H1650 (EGFR mut) Cell Lung >100     2.301 32 2.950 64 53.71 96 A2780 Ovarian 70.90* 1.137 33 2.030 65 5.934 97 A2780CP >100     2.279 34 4.400 66 20.44 98 RXF-393 Renal 96.52* 1.795 35 5.397 67 20.94 99 A498 >100     1.764 36 3.338 68 61.11 100 N87 Gastric 40.62  1.851 37 3.715 69 12.88 101 SiHA Squamous >100*    5.133 38 11.61 70 >100*    102 FaDu >100     1.982 39 2.053 71 21.49 103 DOHH-2 Diffuse Large >100     2.423 40 6.393 72 20.36 104 B-Cell Lymphoma SU-DHL-4 91.30* 1.814 41 3.683 73 11.34 105 SU-DHL-6 50.00* 0.8188 — 3.517 — 10.68 — OCI-LY3 >100*    2.378 42 2.871 74 11.51 106 JIM1 85.16  3.954 43 5.172 75 29.25 107 KHM-1B Human 94.08* 3.365 — 7.575 — 100    — KMM-1 Myeloma 82.16* 4.038 44 5.301 76 30.85 108 KMS-11 >100     4.934 45 4.945 77 68.36 109 KMS-27 91.16* 2.104 46 3.942 78 15.62 110 KMS-34 >100     5.446 47 6.674 79 48.52 111 H929 80.31  4.501 48 4.301 80 >100    112 L363 99.13* 2.003 49 5.271 81 23.05 113 MM.IS >100     1.665 50 2.260 82 14.65 114 MOLP-8 86.44* 1.382 51 2.889 83 14.47 115 Jeko-1 Parental Mantle Cell 89.39* 4.405 52 4.696 84 32.85 116 Jeko-1 Lenalidomide Lymphoma 79.38* 6.193 53 5.070 85 40.79 117 Resistant Jeko-1 Bortezomib 87.62* 4.811 54 4.083 86 31.34 118 Resistant *Value was calculated by averaging using 100 μM for trial value. In cases where one trial value was >100 μM or <0.005 μM, 100 μM or 0.005M was used to average the values and obtain an IC₅₀.

This data demonstrates that the preferred 560, 561, and 562 diharmaline compounds have significantly lower IC₅₀ values as compared with the monoharmaline 518F014 compound. This phenomenon has been found consistent throughout the tested compounds of the invention, namely that the diharmaline compounds are markedly superior as compared with the monoharmaline compounds.

Example 9

In this Example, a compound mixture was prepared by reacting 2:1 by weight harmaline and 3-phenoxybenzaldehyde (3-phenoxybenzaldehyde:harmaline). The reaction mixture had three components, namely fractions having molecular weights of 608 (47% by weight), 788 (32% by weight), and 394 (21% by weight). The MW 608 product contained two moieties of harmaline and one of 3-phenoxybenzaldehyde, with one removed water molecule; the MW 788 product contained two moieties of harmaline and two moieties of 3-phenoxybenzaldehyde, with two removed molecules of water; and the MW 394 product contained one mole each of harmaline and 3-phenoxybenzaldehyde with one water molecule removed. The following table sets forth the results of a series of assays using this compound mixture against 31 different cell lines, where the assays were performed as set forth in Example 2. Two IC₅₀ trials were run in each case, and the results thereof were averaged to give the mean IC₅₀ values.

TABLE 15 594 Trial A Trial B Mean (μg/mL) (μg/mL) (μg/mL) MIA PaCa-2 Pancreatic 3.815 3.169 3.492 ASPC-1 2.941 3.993 3.467 BxPC-3 3.448 3.325 3.387 AN3CA Endometrial 2.793 2.796 2.795 HEC-1a 3.147 3.425 3.286 MDA-MB-231 TNBC 3.099 3.084 3.092 MDA-MB-468 2.898 2.682 2.790 HCC70 6.146 7.590 6.868 H1975 (EGFR mut) NSCLC 2.924 2.867 2.896 H1650 (EGFR mut) 2.606 2.548 2.577 A2780 Ovarian 2.667 2.489 2.578 A2780CP 3.570 2.965 3.268 RXF-393 RCC 3.16 3.161 3.161 A498 2.911 2.995 2.953 N87 Gastric 3.482 5.037 4.260 SiHA Cervical - SCC 8.561 8.894 8.728 FaDu H&N - SCC 3.782 5.453 4.618 DOHH-2 DLBCL 2.577 2.707 2.642 SU-DHL-4 2.769 2.701 2.735 SU-DHL-6 2.480 2.234 2.36 OCI-LY3 3.865 7.536 5.701 JIM1 human 7.414 7.923 7.669 KHM-1B myeloma KMM-1 2.527 2.607 2.567 KMS-11 6.433 5.640 6.037 KMS-27 5.538 2.733 4.136 KMS-34 4.226 3.551 3.889 H929 6.498 5.415 5.957 L363 3.673 3.603 3.638 MM.1S 2.482 2.656 2.569 MOLP-8 2.734 2.703 2.719

Example 10

In this Example, pancreatic cancer cells (S2-007 and Mia-PaCa2) were treated with the previously described compound 518B562 at different times and concentrations, followed by generation of proliferation assays using the techniques described above. This product significantly inhibited the proliferation of the cells in doses of 1-25 μg/mL and in a time-dependent manner of 24-72 hours. After 72 hours of treatment, the IC₅₀ values of the compound against S2-007 and Mia-PaCa2 cells was determined as 3 μg/mL and 5 μg/mL, respectively.

Example 11

In this Example, a clustergram/heat map of RNA sequences for cancer stem cell (CSC) markers was performed before and after treatment of S2-007 human pancreatic cancer cells with 518B562. This experiment was conducted using a Whole Transcriptome Shotgun Sequence (WTSS), followed by bioinformatics data analysis of CSC markers. An RNA-Seq/heat map was generated to obtain a genome-wide gene expression profile of the pancreatic cancer cells.

One sample of the pancreatic cancer cell line was untreated, while an identical sample was treated with 5 micro-g/mL of 518B562. Comparative RNA sequences were performed on the samples using an Illumina HISeq 2500 sequencer at a 100 bp single read resolution. The sequence readings were mapped to the human genome (assembly GRCh38.re177) using the STAR software (Dobin et al. 2012). Transcript abundance estimates were generated using the Cufflinks software (Trapnell et al. 2010) and differential gene expression estimates were calculated using the Cuffdiff software (Trapnell et al. 2013). RNA-Seq generated around 48.6 and 60.1 reads, of which between 97.2% and 98.3% of the reads mapped to the reference genome.

The clustergram or heat map suggested that 518B562 significantly inhibited clusters of genes on proliferative, anti-apoptotic, and angiogenic markers, while up-regulating anti-proliferative and apoptotic markers. Specifically, 518B562 up-regulated apoptotic markers [ICAM5, WNK4, ALPP, LTRC26, SHBG, MT1X] and anti-proliferative markers [NRP1, ATF2A, CYP1b1, ALPP, DEPTOR, MT1F]. Also, 518B562 down-regulated angiogenesis markers [OXTR, SYCP2, CRHR1, SPEG], anti-apoptotic markers [TUG1, FABP1, PI3, DOKS] and proliferation signaling markers [FOXj1, SPP1, C3].

Example 12

6 g of benzaldehyde, 3 g of harmaline, and 50 mL of ethanol were placed in a 250 mL round-bottom flask. This dispersion was refluxed for approximately 4 hours at a temperature of about 78° C., after which it was allowed to cool gradually to room temperature. The resultant solids were collected by vacuum filtration, rinsed with approximately 200 mL of cold water, and dried at room temperature to yield multiply twinned racemic crystalline clumps. A single-domain piece was cut from one of the clumps and gave usable diffraction data, making it possible to locate and refine all of the hydrogen atoms as independent isotropic atoms; two nitrogen atoms also appeared to be protonated. The resultant two-dimensional structure of the 560 compound was determined to be:

The above structure is referred to herein as the “confirmed 560 compound.”

A three-dimensional representation of the above 560 compound is shown below, with the large circles representing carbon atoms, and the small circles representing hydrogen atoms; the double bonds are not shown in this representation. In addition, a hydrogen bond is illustrated in dotted lines between the nitrogens N1 and N4. It is believed that this hydrogen bond may be important to the functionality of the compound. The other numbered atoms are provided for reference.

The above compound may be subject to isomerization, particularly during NMR analysis, to give the following two-dimensional isomeric structure:

A reduced form of either of the above isomers may be less prone to additional isomerization. The two-dimensional structure of this reduced compound (produced by hydrogenation of the above compounds) is set forth below.

Example 13

In this example, several 560 compounds were prepared by mixing together 6 g of benzaldehyde and 3 g of harmaline in closed 40 mL vials, followed by shaking for several minutes. The closed vials were then placed in a water bath at 40° C. for 1-5 days. After cooling, the vials were opened and placed in a Labconco FreeZone 4.5 L freeze dryer at 0.028 kPa and −48° C. for 1 week. The contents of the vials were then mixed with water/methanol combinations, and the resultant solids were collected by vacuum filtration. The molecular weights of the compounds were found to be 302, 320, 514, and 516.

Example 14

6 g of vanillin, 3 g of harmaline, and 50 mL of ethanol were placed in a 250 mL round-bottom flask. This dispersion was refluxed for approximately 4 hours at a temperature of about 78° C., after which it was allowed to cool gradually to room temperature. The resultant solids were collected by vacuum filtration, rinsed with approximately 200 mL of cold water, and dried at room temperature to yield multiply twinned racemic crystalline clumps. A single-domain piece was cut from one of the clumps and gave usable diffraction data, making it possible to locate and refine all of the hydrogen atoms as independent isotropic atoms; two nitrogen atoms also appeared to be protonated. The resultant two-dimensional structure of the 562 compound was determined to be:

Example 15

In this example, the 560 and 562 compounds of Examples 12 and 14, respectively, were tested in cell proliferation assays using two pancreatic cancer cells, S2-007 and MiaCaPa-2. In each assay, 5×10⁴ cells were seeded in 96-well culture plates. After incubation for 24 hours, the cells were treated at various concentrations of the 560 or 562 compounds, and allowed to incubate for further periods of 72 hours. Cell proliferation values were determined by enzymatic hexoseaminidase assay. The results of these tests are set forth in FIGS. 119 and 120 (560 compound), and FIGS. 121 and 122 (562 compound). These Figures also provide the IC50 values for each assay.

Example 16

In this example, the 560 and 562 compounds of Examples 12 and 14, respectively, were tested using cell colony formation assays. 500 viable S2-007 and MiaCaPa-2 cells were plated in six-well dishes and allowed to grow for 24 hours. The cells were then incubated in the presence or absence of the 560 and 562 compounds for 72 hours. The compound-containing media were then removed, and the cells were washed in PBS and incubated for an additional 10 days in complete media. The resultant colonies were then washed in PBS and fixed using 10% formalin for 10 minutes at room temperature, followed by washing with PBS and staining with Crystal Violet. The colonies of the control and compound-supplemented assays were then counted and compared.

FIG. 123 (560 compound, S2-007 cells) illustrates the colony formation with control (no 560 compound), and 4 μg and 6 μg 560 compound at 24, 48, and 72 hours. The 560 compound significantly disrupted colony formation, particularly at the 6 μg level of use.

FIG. 124 (560 compound, MiaPaCa-2 cells) illustrates the colony formation with control (no 560 compound), and 3 μg and 5 μg 560 compound at 24, 48, and 72 hours. The 560 compound significantly disrupted colony formation at both levels of use.

FIG. 125 (562 compound, S2-007 cells) illustrates the colony formation with control (no 562 compound), and 14 μg and 16 μg 562 compound at 24, 48, and 72 hours.

FIG. 126 (562 compound, S2-007 cells) illustrates the colony formation with control (no 562 compound), and 20 μg and 24 μg 562 compound at 24, 48, and 72 hours.

FIG. 127 (562 compound, MiaPaCa-2 cells) illustrates the colony formation with control (no 562 compound), and 3 μg, 7 μg, 10 μg, and 12 μg 562 compound at 24, 48, and 72 hours. The 562 compound significantly disrupted colony formation, particularly at the higher levels of use.

Example 17

In this example, the 560 and 562 compounds of Examples 12 and 14, respectively were used in cell cycle assays against S2-07 and MiaPaCa02 cells. In each instance, cells treated with the 560 and 562 compounds for 72 hours were trypsinized and suspended in PBS. The single-cell suspensions were fixed using pre-chilled 70% ethanol for 3 hours, and were subsequently permeabilized with PBS containing 0.1% Triton X-100, 1 mg/mL propidium iodide, and 2 mg DNase-free RNase at room temperature. Flow cytometry assays were then performed using a FASCalibur analyzer (Becton Dickinson), capturing 10,000 events for each sample. The results were analyzed using ModFit LT™ software (Verity Software House). These results were carried out using a Sub G0 gating window, alternately open (with Sub G0) and closed (without Sub G0), to give quiescent state and active state cell data.

FIGS. 128 and 128A (compound 560 S2-007 cells) illustrates the cell cycle results at 24 and 48 hours with and without Sub G0.

FIGS. 129 and 129A (compound 560 MiaPaCa-2 cells) illustrates the cell cycle results at 24, 48, and 72 hours with and without Sub G0.

FIGS. 130 and 130A (compound 562 S2-007 cells) illustrates the cell cycle results at 24, 48, and 72 hours with and without Sub G0.

FIGS. 131 and 131A (compound 562 MiaPaCa-2 cells) illustrates the cell cycle results at 24, 48, and 72 hours with and without Sub G0.

Example 18

2200 mg of benzaldehyde plus 1100 mg harmaline were placed in a 50 mL beaker and mixed slightly. The beaker was heated for several minutes until a color change was observed, whereupon the mixture was transferred to a 250 mL round-bottom flask with the aid of 50 mL of isopropyl alcohol. Next, 50 μL of 37% HCl was added, and the mixture was refluxed for 1.5 hours. After about 1 hour, crystals began to form. After refluxing, the reaction mixture was allowed to sit and cool to ambient temperature, and was filtered using a Buchner funnel, with cold isopropyl alcohol rinsing. The weight of the collected product was about 700 mg, which was beet red in color. Analysis of the compound indicated that it contained approximately 18% of the confirmed 560 compound (mw=516), and approximately 80% of a dimer (mw=604).

Example 19

3000 mg of benzaldehyde plus 1500 mg harmaline were placed in a 50 mL beaker and mixed slightly. The beaker was heated until the color of the mixture changed to light brown. The mixture was then transferred to a 250 mL round-bottom flask with the aid of 30 mL of isopropyl alcohol. Next, 50 μL of 37% HCl was added, and the mixture was refluxed for 30 minutes. After refluxing, the reaction mixture was allowed to sit and cool to ambient temperature, and was filtered using a Buchner funnel, with cold isopropyl alcohol rinsing. The weight of the collected product was about 1325 mg, which was yellow in color. Analysis of the compound indicated that it contained approximately 80% of the confirmed 560 compound (mw=516).

Example 20

8000 mg of benzaldehyde plus 4000 mg harmaline were placed in a 50 mL beaker and mixed slightly. The mixture was then transferred to a 250 mL round-bottom flask with the aid of 40 mL of methyl alcohol. Next, 50 μL of 37% HCl was added, and the mixture was refluxed for 45 minutes. After refluxing, the reaction mixture was allowed to sit and cool overnight. After cooling, the mixture was filtered using a Buchner funnel, with methyl alcohol rinsing. The product was yellow in color. Analysis of the compound indicated that it contained approximately 93% of the confirmed 560 compound (mw=516), the remainder being unreacted harmaline.

Example 21

8000 mg of vanillin plus 4000 mg harmaline were placed in a 50 mL beaker. The mixture was heated by application of 40° C. water to the outside of the beaker, which initiated a reaction and caused the mixture to change from yellow to light brown in color. The mixture was then transferred to a 250 mL round-bottom flask with the aid of 50 mL of isopropyl alcohol, causing the mixture to turn to a yellow-green color. Once all of the reactants were solubilized, the color changed from yellow-green to yellow-brown. Next, 150 μL of 37% HCl was added, with heating until the mixture turned dark brown in color and produced a bluish precipitate. The mixture was then refluxed for 25 minutes, and the vessel was cooled using tap water. The reaction mixture was then filtered using a Buchner funnel and rinsed with isopropyl alcohol. Thereupon, the mixture was oven-dried, producing an ashy grey-blue color. Analysis of the compound indicated that it contained approximately 85% of the Example 14 562 compound (mw=562).

Example 22

4000 mg of vanillin plus 2000 mg harmaline were placed in a 50 mL beaker. The mixture was heated by application of 40° C. water to the outside of the beaker, which initiated a reaction and caused the mixture to change from yellow to dark brown in color. The mixture was then transferred to a 250 mL round-bottom flask with the aid of 50 mL of isopropyl alcohol. Once all of the reactants were solubilized, the color changed to dark red-brown. Next, 500 μL of 37% HCl was added, with heating until the mixture turned dark brown in color and produced a bluish precipitate. The mixture was then refluxed for 45 minutes, and the vessel was cooled using tap water. The reaction mixture was then filtered using a Buchner funnel and rinsed with isopropyl alcohol. Thereupon, the mixture was oven-dried, to yield approximately 1500 mg of dark brown product. Analysis of the compound indicated that it contained approximately 60% of the Example 14 562 compound (mw=562), about 23% dehydrated adduct (mw=348), and about 17% dimer (mw=696).

Example 23

2 g of cuminaldehyde, 1 g of harmaline, and 40 mL of ethanol were placed in a 250 mL round-bottom flask. This dispersion was refluxed for approximately 30 minutes at a temperature of about 65° C., after which the mixture was allowed to cool gradually overnight to room temperature. The resultant bottom liquid products were then analyzed. The principal compound, referred to herein as the 561 product, has the following structure:

Example 24

In this test, a related diharmaline compound 594, diharmaline 3-phenoxybenzaldehyde was tested using the same cell lines and procedures as described in Example 8. The structure of the 594 compound is set forth below, and the collected cell proliferation assay data is given in the following table.

TABLE 16 Trial A Trial B Mean Cell line Tissue Type (μg/mL) (μg/mL) (μg/mL) MIA Pancreatic 3.815 3.169 3.492 PaCa-2 ASPC-1 2.941 3.993 3.467 BxPC-3 3.448 3.325 3.387 AN3CA Endometrial 2.793 2.796 2.795 HEC-1a 3.147 3.425 3.286 MDA-MB-231 Triple 3.099 3.084 3.092 MDA-MB-468 Negative 2.898 2.682 2.790 HCC70 Breast 6.146 7.590 6.868 H1975 (EGFR mut) Non Small 2.924 2.867 2.896 H1650 (EGFR mut) Cell Lung 2.606 2.548 2.577 A2780 Ovarian 2.667 2.489 2.578 A2780CP 3.570 2.965 3.268 RXF-393 RCC 3.16 3.161 3.161 A498 4.817 4.561 4.689 N87 Gastric 3.482 5.037 4.260 SiHA Squamous 8.561 8.894 8.728 FaDu 3.782 5.453 4.618 DOHH-2 Diffuse 2.577 2.707 2.642 SU-DHL-4 Large B-Cell 2.769 2.701 2.735 SU-DHL-6 Lymphoma 2.480 2.234 2.357 OCI-LY3 3.865 7.536 5.701 JIM1 human 7.414 7.923 7.669 KHM-1B myeloma 6.483 6.810 6.647 KMM-1 2.527 2.607 2.567 KMS-11 6.433 5.640 6.037 KMS-27 5.538 2.733 4.136 KMS-34 4.226 3.551 3.889 H929 6.498 5.415 5.957 L363 3.673 3.603 3.638 MM.1S 2.482 2.656 2.569 MOLP-8 2.734 2.703 2.719

As evidenced by the above data, the 594 compound is effective against a wide variety of cancer cells.

Example 25

In this example, different cell lines were subjected to in vitro cell proliferation assays as described in Example 8. In particular, the cell lines are identified in the following Table 17, and the tissue types were: Pancreatic; Endometrial; Triple-Negative Breast Cancer (TNBC); Non-Small-Cell Lung Carcinoma (NSCLC); Ovarian; Renal Cell Carcinoma (RCC); Cervical-Squamous Cell; Hemagglutinin and Neuraminidase (H&N-Squamous Cell); Germinal Center B Cell-Like Novo Diffuse Large B-Cell Lymphoma (GCB-DLBCL); Activated B-Cell—Diffuse Large B Cell Lymphoma (ABC-DLBCL); Human Myeloma; Human Myeloma Cell Line Lymphocyte-Like; Human Myeloma Cell Line Lymphocyte-Like Myeloma Pleural Effusion Infiltration; Human Multiple Myeloma; Plasma Cell Leukemia/Multiple Myeloma Epstein-Barr Nuclear Antigen-Negative (EBNA-Negative) and to Express mRNA for Proto-Oncogene B-Cell Lymphoma 2 (BCL2); Multiple Myeloma from Peripheral Blood Type IgD Lmabda.

These cell lines were tested with a series of diharmaline/aldehyde compounds in accordance with the invention. The aldehydes reacted with harmaline are listed in Table 17 as nos. 1-2, 4-15, 17-24, and 27-30, and the similarly numbered corresponding compounds are set forth after Table 17. See the Key below for details. The compounds are identified in the section following Table 17. In each case, the compounds were prepared by reacting overnight one part by weight harmaline and two parts by weight aldehyde in ethanol at 50° C.

Key - Tables 17-19 * Value was averaged using 100 μM trial value HMCL Human myeloma cell line HMCL-A Human myeloma cell line lymphocyte-like HMCL-B Human myeloma cell line lymphocyte-like myeloma pleural effusion infiltration (IgAk) HMM Human Multiple Myeloma MMIgD Multiple myeloma from peripheral blood type IgD lmabda PCL/MM Plasma cell leukemia/multiple myeloma EBNA-negative and to express mRNA for proto-oncogene BCL2 SC Squamous Cell

TABLE 17 Tissue Type Mean IC₅₀ (μM) Compounds Cell Line (see Key) 1 2 4 5 6 7 MIA PaCa-2 Pancreatic 3.67 7.51 4.37 19.94 13.64 16.21 ASPC-1 Pancreatic 6.44 15.25 10.99 15.73 100*   40.91 BxPC-3 Pancreatic 4.30 7.88 6.05 21.24 15.82 12.32 AN3CA Endometrial 2.50 6.72 5.10 11.39 21.45 11.46 HEC-1a Endometrial 9.27 21.61 16.26 26.98 100*   71.10 MDA-MB-231 TNBC 5.72 14.35 11.99 23.73 60.51 31.70 MDA-MB-468 TNBC 4.39 9.90 6.27 14.73 14.54 11.18 HCC70 TNBC 8.02 21.18 11.55 15.09 100*   23.45 H1975 (EGFR mut) NSCLC 6.54 19.49 14.09 28.11 100*   60.83 H1650 (EGFR mut) NSCLC 8.18 21.60 13.60 23.40 100*   64.17 A2780 Ovarian 5.90 16.95 11.50 24.12 100*   32.96 A2780CP Ovarian 4.45 20.22 10.78 24.62 70.66 33.68 A498 RCC 9.91 16.76 12.71 22.76 100*   29.34 SiHA Cervical - SC 9.44 17.07 17.09 36.85 74.79 48.07 FaDu H&N - SC 5.36 13.54 7.69 22.26 15.76 13.70 DoHH-2 GCB-DLBCL 2.25 3.66 3.17 9.31  6.91 6.48 OCI-LY3 ABC-DLBCL 3.42 6.58 5.57 12.35 11.30 11.42 JIM1 HMCL 4.51 11.54 6.89 17.73 18.00 14.41 KMM-1 HMCL-A 4.20 9.40 6.32 20.33 16.89 19.89 KMS-34 HMCL-B 4.15 9.35 4.75 17.40 10.97 11.13 RPMI-8226 HMM 1.87 8.51 4.09 19.31 10.35 11.79 L363 PCL/MM 2.62 6.26 6.38 22.15 16.33 11.75 MOLP-8 MM IgD 1.98 5.30 5.03 14.44 11.48 11.74 Tissue Type Mean IC₅₀ (μM) Compounds Cell Line (see Key) 8 9 10 11 12 MIA PaCa-2 Pancreatic 24.16 52.61 11.88 11.67 11.66 ASPC-1 Pancreatic 55.59 69.99 23.76 21.93 31.31 BxPC-3 Pancreatic 21.95 30.00 27.62 11.53 11.26 AN3CA Endometrial 20.21 29.33 31.66 8.74 5.36 HEC-1a Endometrial 65.84 95.60 88.11 36.27 24.84 MDA-MB-231 TNBC 24.35 45.82 43.67 13.47 11.35 MDA-MB-468 TNBC  8.45 12.69 11.39 12.20 11.64 HCC70 TNBC 24.92 38.95 50.24 27.23 22.03 H1975 (EGFR mut) NSCLC 55.14 54.62 64.03 19.47 13.00 H1650 (EGFR mut) NSCLC 59.92 79.70 13.27 26.18 21.58 A2780 Ovarian 18.72 35.66 65.05 18.30 21.25 A2780CP Ovarian 49.81 60.32 29.41 24.87 20.47 A498 RCC 78.66 100*    79.22* 51.27 40.94 SiHA Cervical - SC 100*   100*   100*   15.86 50.19 FaDu H&N - SC 16.11  72.34* 49.71 16.45 18.20 DoHH-2 GCB-DLBCL  6.37  9.76  2.78 4.32 3.72 OCI-LY3 ABC-DLBCL 11.34 11.94  7.57 11.34 11.29 JIM1 HMCL 14.29 40.60 20.09 11.53 6.04 KMM-1 HMCL-A 13.23 16.01 11.08 12.05 10.61 KMS-34 HMCL-B 11.05 11.37  4.87 11.14 7.19 RPMI-8226 HMM 11.51 11.52  3.98 10.11 10.17 L363 PCL/MM 12.59 19.73 35.62 11.58 10.91 MOLP-8 MM IgD 10.09  6.59  8.28 10.75 3.88 Tissue Type Mean IC₅₀ (μM) Compounds Cell Line (see Key) 13 14 15 17 18 MIA PaCa-2 Pancreatic 15.82 22.17 3.63 29.05 32.49 ASPC-1 Pancreatic 82.50 53.09 5.45 37.85 43.78 BxPC-3 Pancreatic 16.55 21.03 4.22 23.27 36.19 AN3CA Endometrial  7.82 9.05 3.45 29.72 28.80 HEC-1a Endometrial 17.43 28.71 13.14 46.90 49.81 MDA-MB-231 TNBC 25.81 32.39 6.77 56.18 43.88 MDA-MB-468 TNBC 12.62 16.85 6.70 42.23 48.66 HCC70 TNBC 28.51 33.33 11.67 45.41 36.28 H1975 (EGFR mut) NSCLC 14.03 33.61 12.09 44.48 59.06 H1650 (EGFR mut) NSCLC 21.20 25.45 9.60 48.68 45.40 A2780 Ovarian 11.76 21.24 8.45 40.55 26.34 A2780CP Ovarian 23.45 24.27 7.63 39.07 31.23 A498 RCC  91.59* 54.70 15.24 89.27  93.43* SiHA Cervical - SC 100*   51.89 12.34 100*   100*   FaDu H&N-SC 13.48 19.18 6.62 42.89 49.24 DoHH-2 GCB-DLBCL  6.52 7.90 5.05 12.07  8.55 OCI-LY3 ABC-DLBCL 11.69 15.39 6.32 33.77 24.97 JIM1 HMCL 11.53 17.90 6.26 37.17 39.83 KMM-1 HMCL-A 13.11 23.94 7.36 37.72 33.58 KMS-34 HMCL-B 11.12 14.64 3.50 12.49 17.80 RPMI-8226 HMM  9.10 7.53 2.95 11.97 12.27 L363 PCL/MM 12.93 23.40 6.58 46.74 34.78 MOLP-8 MM IgD 10.99 10.07 3.19 16.98 12.25 Tissue Type Mean IC₅₀ (μM) Compounds Cell Line (see Key) 19 20 21 22 23 MIA PaCa-2 Pancreatic 36.62 2.72 4.41 5.93 5.64 ASPC-1 Pancreatic 45.46 3.66 5.11 8.61 9.26 BxPC-3 Pancreatic 36.21 2.25 3.58 5.37 4.97 AN3CA Endometrial 22.78 1.86 3.93 4.15 2.38 HEC-1a Endometrial 45.04 10.99 11.67 11.78 12.52 MDA-MB-231 TNBC 41.75 4.71 5.95 7.12 5.94 MDA-MB-468 TNBC 48.89 5.12 6.74 11.45 6.54 HCC70 TNBC 50.90 6.35 10.86 13.18 10.87 H1975 (EGFR mut) NSCLC 54.97 6.41 11.36 11.49 8.23 H1650 (EGFR mut) NSCLC 56.84 4.44 8.22 11.12 5.89 A2780 Ovarian 22.06 3.75 7.51 9.16 5.07 A2780CP Ovarian 27.51 5.06 9.87 9.30 6.49 A498 RCC 89.50 11.28 11.69 21.57 21.44 SiHA Cervical - SC 75.94 6.73 11.46 11.68 11.66 FaDu H&N - SC 44.44 4.05 6.26 11.09 6.14 DoHH-2 GCB-DLBCL 10.22 1.88 3.61 4.26 3.73 OCI-LY3 ABC-DLBCL 20.56 3.85 4.94 9.73 5.45 JIM1 HMCL 42.85 4.28 5.55 7.35 6.32 KMM-1 HMCL-A 25.60 3.66 6.41 6.21 3.98 KMS-34 HMCL-B 31.17 3.26 3.82 3.90 3.47 RPMI-8226 HMM 10.58 1.27 3.56 2.06 1.28 L363 PCL/MM 36.82 3.89 5.15 6.12 4.72 MOLP-8 MM IgD 9.81 2.28 3.69 3.83 3.19 Tissue Type Mean IC₅₀ (μM) Compounds Cell Line (see Key) 24 27 28 29 30 MIA PaCa-2 Pancreatic 12.98 5.83 4.51 11.27 5.47 ASPC-1 Pancreatic 32.67 7.34 7.22 10.77 10.97 BxPC-3 Pancreatic 10.76 4.61 6.58 11.46 7.73 AN3CA Endometrial 11.23 4.36 4.49  8.07 5.01 HEC-1a Endometrial 48.18 11.65 10.55  100*   9.91 MDA-MB-231 TNBC 20.67 7.39 10.14  13.94 11.06 MDA-MB-468 TNBC 16.55 10.76 8.43 100*   7.46 HCC70 TNBC 25.74 11.37 11.92  39.98 18.53 H1975 (EGFR mut) NSCLC 11.66 11.50 100*    13.64 11.39 H1650 (EGFR mut) NSCLC 12.48 11.34 11.34  13.88 16.58 A2780 Ovarian 10.62 8.09 100*    12.11 6.45 A2780CP Ovarian 17.80 11.10 7.02 11.45 8.55 A498 RCC 83.81* 11.74 16.31  42.53 17.11 SiHA Cervical - SC 58.90 11.60 13.32  46.86 17.76 FaDu H&N-SC 11.68 11.15 5.64 11.41 6.82 DoHH-2 GCB-DLBCL 5.58 2.52 2.50  2.09 5.36 OCI-LY3 ABC-DLBCL 11.43 9.27 6.17  9.63 7.28 JIM1 HMCL 56.09 11.36 8.08 16.58 7.07 KMM-1 HMCL-A 11.26 7.18 6.43  7.71 5.81 KMS-34 HMCL-B 7.84 4.44 4.44  4.40 4.81 RPMI-8226 HMM 5.05 3.21 3.50  4.44 4.30 L363 PCL/MM 13.19 7.83 4.75 11.43 5.57 MOLP-8 MM IgD 10.57 3.97 3.71  5.89 2.70

Compounds:

Example 26

In this Example, a compound was produced via the reaction between a fused bicyclic compound, namely 1-methyl-3,4-dihydroisoquinoline and vanillin, as described in Example 25. This compound was tested against the same cell lines of Example 25, giving the following results.

TABLE 18 Tissue Type Mean IC₅₀ Cell Line (see Key) (μM)SAR02 MIA PaCa-2 Pancreatic 14.67 ASPC-1 Pancreatic 18.68 BxPC-3 Pancreatic 10.74 AN3CA Endometrial 14.16 HEC-1a Endometrial 23.35 MDA-MB-231 TNBC 22.02 MDA-MB-468 TNBC 13.23 HCC70 TNBC 27.82 H1975 (EGFR mut) NSCLC 17.88 H1650 (EGFR mut) NSCLC 8.85 A2780 Ovarian 10.41 A2780CP Ovarian 23.82 A498 RCC 31.12 SiHA Cervical - SC 35.90 FaDu H&N - SC 14.30 DoHH-2 GCB-DLBCL 10.06 OCI-LY3 ABC-DLBCL 7.28 JIM1 HMCL 14.97 KMM-1 HMCL-A 12.32 KMS-34 HMCL-B 5.31 RPMI-8226 HMM 6.06 L363 PCL/MM 12.21 MOLP-8 MM IgD 9.82

Example 27

In this Example, 200 mg of 4-methyl-6,7-dihydroithienol[3,2-c]pyridine of the formula

was reacted with an excess of vanillin in methanol at 100° C. by microwaving the reaction mixture for 30 minutes. Unexpectedly, a spirocyclic solid compound was recovered having the formula

which was the MW 588.82 species hydrogen bonded with the MW 151.14 species. Note that the MW 588.82 species comprised a single vanillin moiety with three moieties of 4-methyl-6,7-dihydroithienol[3,2-c]pyridine. The spirocyclic compound (designated HRM 05) was tested by the above in vitro cell proliferation assay against a number of different tissue types, with the following results:

TABLE 19 Tissue Type Mean IC₅₀ Cell Line (see Key) (μM)HRM05 MIA PaCa-2 Pancreatic 12.20 ASPC-1 Pancreatic 20.22 BxPC-3 Pancreatic TBD AN3CA Endometrial TBD HEC-1a Endometrial TBD MDA-MB-231 TNBC TBD MDA-MB-468 TNBC TBD HCC70 TNBC TBD H1975 (EGFR mut) NSCLC TBD H1650 (EGFR mut) NSCLC TBD A2780 Ovarian 15.86 A2780CP Ovarian TBD A498 RCC TBD SiHA Cervical - SC TBD FaDu H&N - SC 13.97 DoHH-2 GCB-DLBCL 17.59 OCI-LY3 ABC-DLBCL TBD JIM1 HMCL 17.58 KMM-1 HMCL-A 12.45 KMS-34 HMCL-B TBD RPMI-8226 HMM TBD L363 PCL/MM 13.66 MOLP-8 MM IgD TBD TBD = To Be Determined

While the anti-cancer properties of the compositions of the invention have been demonstrated against certain cancers, it is considered that the invention may be applicable to virtually all cancers, such as the following: Acute Lymphoblastic Leukemia, Adult; Acute Lymphoblastic Leukemia, Childhood; Acute Myeloid Leukemia, Adult; Acute Myeloid Leukemia, Childhood; Adrenocortical Carcinoma; Adrenocortical Carcinoma, Childhood; Adolescents, Cancer in; AIDS-Related Cancers; AIDS-Related Lymphoma; Anal Cancer; Appendix Cancer; Astrocytomas, Childhood; Atypical Teratoid/Rhabdoid Tumor, Childhood, Central Nervous System; Basal Cell Carcinoma; Bile Duct Cancer, Extrahepatic; Bladder Cancer; Bladder Cancer, Childhood; Bone Cancer, Osteosarcoma and Malignant Fibrous Histiocytoma; Brain Stem Glioma, Childhood; Brain Tumor, Adult; Brain Tumor, Brain Stem Glioma, Childhood; Brain Tumor, Central Nervous System Atypical Teratoid/Rhabdoid Tumor, Childhood; Brain Tumor, Central Nervous System Embryonal Tumors, Childhood; Brain Tumor, Astrocytomas, Childhood; Brain Tumor, Craniopharyngioma, Childhood; Brain Tumor, Ependymoblastoma, Childhood; Brain Tumor, Ependymoma, Childhood; Brain Tumor, Medulloblastoma, Childhood; Brain Tumor, Medulloepithelioma, Childhood; Brain Tumor, Pineal Parenchymal Tumors of Intermediate Differentiation, Childhood; Brain Tumor, Supratentorial Primitive Neuroectodermal Tumors and Pineoblastoma, Childhood; Brain and Spinal Cord Tumors, Childhood (Other); Breast Cancer; Breast Cancer and Pregnancy; Breast Cancer, Childhood; Breast Cancer, Male; Bronchial Tumors, Childhood; Burkitt Lymphoma; Carcinoid Tumor, Childhood; Carcinoid Tumor, Gastrointestinal; Carcinoma of Unknown Primary; Central Nervous System Atypical Teratoid/Rhabdoid Tumor, Childhood; Central Nervous System Embryonal Tumors, Childhood; Central Nervous System (CNS) Lymphoma, Primary; Cervical Cancer; Cervical Cancer, Childhood; Childhood Cancers; Chordoma, Childhood; Chronic Lymphocytic Leukemia; Chronic Myelogenous Leukemia; Chronic Myeloproliferative Disorders; Colon Cancer; Colorectal Cancer, Childhood; Craniopharyngioma, Childhood; Cutaneous T-Cell Lymphoma; Embryonal Tumors, Central Nervous System, Childhood; Endometrial Cancer; Ependymoblastoma, Childhood; Ependymoma, Childhood; Esophageal Cancer; Esophageal Cancer, Childhood; Esthesioneuroblastoma, Childhood; Ewing Sarcoma Family of Tumors; Extracranial Germ Cell Tumor, Childhood; Extragonadal Germ Cell Tumor; Extrahepatic Bile Duct Cancer; Eye Cancer, Intraocular Melanoma; Eye Cancer, Retinoblastoma; Gallbladder Cancer; Gastric (Stomach) Cancer; Gastric (Stomach) Cancer, Childhood; Gastrointestinal Carcinoid Tumor; Gastrointestinal Stromal Tumor (GIST); Gastrointestinal Stromal Cell Tumor, Childhood; Germ Cell Tumor, Extracranial, Childhood; Germ Cell Tumor, Extragonadal; Germ Cell Tumor, Ovarian; Gestational Trophoblastic Tumor; Glioma, Adult; Glioma, Childhood Brain Stem; Hairy Cell Leukemia; Head and Neck Cancer; Heart Cancer, Childhood; Hepatocellular (Liver) Cancer, Adult (Primary); Hepatocellular (Liver) Cancer, Childhood (Primary); Histiocytosis, Langerhans Cell; Hodgkin Lymphoma, Adult; Hodgkin Lymphoma, Childhood; Hypopharyngeal Cancer; Intraocular Melanoma; Islet Cell Tumors (Endocrine Pancreas); Kaposi Sarcoma; Kidney (Renal Cell) Cancer; Kidney Cancer, Childhood; Langerhans Cell Histiocytosis; Laryngeal Cancer; Laryngeal Cancer, Childhood; Leukemia, Acute Lymphoblastic, Adult; Leukemia, Acute Lymphoblastic, Childhood; Leukemia, Acute Myeloid, Adult; Leukemia, Acute Myeloid, Childhood; Leukemia, Chronic Lymphocytic; Leukemia, Chronic Myelogenous; Leukemia, Hairy Cell; Lip and Oral Cavity Cancer; Liver Cancer, Adult (Primary); Liver Cancer, Childhood (Primary); Lung Cancer, Non-Small Cell; Lung Cancer, Small Cell; Lymphoma, AIDS-Related; Lymphoma, Burkitt; Lymphoma, Cutaneous T-Cell; Lymphoma, Hodgkin, Adult; Lymphoma, Hodgkin, Childhood; Lymphoma, Non-Hodgkin, Adult; Lymphoma, Non-Hodgkin, Childhood; Lymphoma, Primary Central Nervous System (CNS); Macroglobulinemia, Waldenstrom; Malignant Fibrous Histiocytoma of Bone and Osteosarcoma; Medulloblastoma, Childhood; Medulloepithelioma, Childhood; Melanoma; Melanoma, Intraocular (Eye); Merkel Cell Carcinoma; Mesothelioma, Adult Malignant; Mesothelioma, Childhood; Metastatic Squamous Neck Cancer with Occult Primary; Mouth Cancer; Multiple Endocrine Neoplasia Syndromes, Childhood; Multiple Myeloma/Plasma Cell Neoplasm; Mycosis Fungoides; Myelodysplastic Syndromes; Myelodysplastic/Myeloproliferative Neoplasms; Myelogenous Leukemia, Chronic; Myeloid Leukemia, Adult Acute; Myeloid Leukemia, Childhood Acute; Myeloma, Multiple; Myeloproliferative Disorders, Chronic; Nasal Cavity and Paranasal Sinus Cancer; Nasopharyngeal Cancer; Nasopharyngeal Cancer, Childhood; Neuroblastoma; Non-Hodgkin Lymphoma, Adult; Non-Hodgkin Lymphoma, Childhood; Non-Small Cell Lung Cancer; Oral Cancer, Childhood; Oral Cavity Cancer, Lip and; Oropharyngeal Cancer; Osteosarcoma and Malignant Fibrous Histiocytoma of Bone; Ovarian Cancer, Childhood; Ovarian Epithelial Cancer; Ovarian Germ Cell Tumor; Ovarian Low Malignant Potential Tumor; Pancreatic Cancer; Pancreatic Cancer, Childhood; Pancreatic Cancer, Islet Cell Tumors; Papillomatosis, Childhood; Paranasal Sinus and Nasal Cavity Cancer; Parathyroid Cancer; Penile Cancer; Pharyngeal Cancer; Pineal Parenchymal Tumors of Intermediate Differentiation, Childhood; Pineoblastoma and Supratentorial Primitive Neuroectodermal Tumors, Childhood; Pituitary Tumor; Plasma Cell Neoplasm/Multiple Myeloma; Pleuropulmonary Blastoma, Childhood; Pregnancy and Breast Cancer; Primary Central Nervous System (CNS) Lymphoma; Prostate Cancer; Rectal Cancer; Renal Cell (Kidney) Cancer; Renal Pelvis and Ureter, Transitional Cell Cancer; Respiratory Tract Cancer with Chromosome 15 Changes; Retinoblastoma; Rhabdomyosarcoma, Childhood; Salivary Gland Cancer; Salivary Gland Cancer, Childhood; Sarcoma, Ewing Sarcoma Family of Tumors; Sarcoma, Kaposi; Sarcoma, Soft Tissue, Adult; Sarcoma, Soft Tissue, Childhood; Sarcoma, Uterine; Sézary Syndrome; Skin Cancer (Nonmelanoma); Skin Cancer, Childhood; Skin Cancer (Melanoma); Skin Carcinoma, Merkel Cell; Small Cell Lung Cancer; Small Intestine Cancer; Soft Tissue Sarcoma, Adult; Soft Tissue Sarcoma, Childhood; Squamous Cell Carcinoma; Squamous Neck Cancer with Occult Primary, Metastatic; Stomach (Gastric) Cancer; Stomach (Gastric) Cancer, Childhood; Supratentorial Primitive Neuroectodermal Tumors, Childhood; T-Cell Lymphoma, Cutaneous; Testicular Cancer; Testicular Cancer, Childhood; Throat Cancer; Thymoma and Thymic Carcinoma; Thymoma and Thymic Carcinoma, Childhood; Thyroid Cancer; Thyroid Cancer, Childhood; Transitional Cell Cancer of the Renal Pelvis and Ureter; Trophoblastic Tumor, Gestational; Unknown Primary Site, Carcinoma of, Adult; Unknown Primary Site, Cancer of, Childhood; Unusual Cancers of Childhood; Ureter and Renal Pelvis, Transitional Cell Cancer; Urethral Cancer; Uterine Cancer, Endometrial; Uterine Sarcoma; Vaginal Cancer; Vaginal Cancer, Childhood; Vulvar Cancer; Waldenström Macroglobulinemia; Wilms Tumor; Women's Cancers. 

We claim:
 1. An anti-cancer composition comprising a therapeutic compound having a pair of fused tricyclic moieties bound together by a single linker moiety, where at least one of the rings of each of the fused tricyclic moieties is a N-containing ring, said linker moiety providing bonding branches from a single non-metal atom forming at least a part of the linker moiety, such that said fused tricyclic moieties are bonded to said linker through said single atom, said linker including a methine group, said single non-metal atom being the carbon atom of said methine group.
 2. The composition of claim 1, said composition including another ingredient selected from the group consisting of active agents, preservatives, buffering agents, salts, carriers, excipients, diluents, and other pharmaceutically acceptable ingredients, and combinations thereof.
 3. The composition of claim 1, said at least one N-containing ring of each fused tricyclic moiety being a terminal ring thereof.
 4. The composition of claim 3, said bonding branches bonded to said terminal N-containing rings of the fused tricyclic moieties.
 5. The composition of claim 3, said terminal ring of each fused tricyclic moiety being a six-membered ring.
 6. The composition of claim 3, said bonding branches bonded to a carbon atom in an alpha position adjacent the N atom of said N-containing rings of the fused tricyclic moieties.
 7. The composition of claim 1, said linker moiety derived from an aldehyde compound.
 8. The composition of claim 7, said linker being an aldehyde moiety selected from the group consisting of moieties of vanillin, benzaldehyde, cinnamaldehyde, cuminaldehyde, orthovanillin, vanillin isobutyrate, phenoxy benzaldehyde, and mixtures thereof.
 9. The composition of claim 7, said linker moiety derived from a compound having the structure

where the substituents may be located at any position around the ring, R1′ is a C1-C12 aldehyde, R2′-R5′ are independently and selectively taken from the group consisting of H, OH, C1-C12 alkyl groups, C2-C12 alkenyl groups, C1-C12 alkoxy groups, C1-C12 aldehyde groups, acetate, isobutyrate, phenyl, phenoxy, benzyloxy, C2-C6 alkyl esters, halo, and nitro, where the dotted bond lines in the six-membered ring represent that the six-membered ring may be cyclohexane, or have one, two, or three carbon-carbon double bonds, or a C1-C12 alkyl or C2-C12 alkenyl aldehyde, said single atom being the carbonyl carbon of said aldehyde group.
 10. The composition of claim 1, said tricyclic moieties each being β-carboline moieties, where each of the β-carboline moieties is independently selected and derived from compounds having the structure

where the numbered 6-member fused ring is a N-heterocycle with a single N atom at any of the positions 2-5, and the R6 substituents may be located at any ring position, R5′ is H or C1-C12 alkoxy, and R6′ is H, a C1-C12 alkyl, or a C1-C12 carboxylic acid.
 11. The composition of claim 1, said composition being in a form selected from the group consisting of liquid dispersions, capsules, pills, tablets, and combinations of any of the foregoing.
 12. The composition of claim 1, said therapeutic compound having two harmaline moieties and a single linker moiety derived from an aldehyde compound.
 13. The composition of claim 12, said aldehyde moiety selected from the group consisting of moieties of vanillin, benzaldehyde, cinnamaldehyde, cuminaldehyde, orthovanillin, vanillin isobutyrate, phenoxy benzaldehyde, and mixtures thereof.
 14. The composition of claim 1, said therapeutic compound having two harmaline moieties and a cinnamaldehyde moiety, and having a molecular weight of approximately
 542. 15. The composition of claim 1, said therapeutic compound having the structure

where the —O—R3 groups may be independently located at any position on the terminal phenyl groups, where each R1 is independently selected from the group consisting of H, OH, and C1-C12 alkyl groups, each R2 is independently selected form the group consisting of H, OH, and C1-C12 alkyl groups, each R3 group is independently selected from the group consisting of C1-C12 alkyl groups, and substituted or unsubstituted phenyl groups, and wherein the designation

refers to the fact that there may optionally be: 1) one or two non-fused double bonds at one or two valence-permitted positions around either or both of the six-membered, N-containing rings; 2) a double bond between either or both of the N-containing rings and the adjacent carbons of the central moiety, with or without an additional non-fused double bond at any valence-permitted position around the corresponding N-containing ring; or 3) either or both of the N-containing rings are free of non-fused double bonds and each R1 is independently selected from the group consisting of H, OH, and C1-C12 alkyl groups.
 16. The composition of claim 15, said structure being


17. The composition of claim 1, said therapeutic compound comprising two harmaline moieties and one vanillin moiety.
 18. The composition of claim 1, said therapeutic compound having the structure

where each R4 is independently selected from the group consisting of nothing, H, OH, and C1-C12 alkyl groups, each R5 is independently selected from the group consisting of H, OH, and C1-C12 alkyl groups, each R6 group is independently located at any position around the corresponding terminal phenyl group, or at either of the two open positions of the two N-containing rings, and is selected from the group consisting of C1-C12 alkoxy groups, H, OH, and substituted or unsubstituted phenyl groups, R7 and R8 are attached at any position around the benzene ring and are independently selected from the group consisting of H, OH, and C1-C12 alkoxy groups, with the proviso that R7 and R8 are not both H, R7 is OH, R8 is a C1-C12 alkoxy group, and each R9 is independently selected from the group consisting of H, OH, and C1-C12 alkyl groups, and wherein the designation

refers to the fact that there may optionally be: 1) zero, one, or two non-fused double bonds at one or two valence-permitted positions around either or both of the six-membered, N-containing rings; 2) a double bond between either or both of the N-containing rings and the adjacent carbons of the central moiety, with or without an additional non-fused double bond at any valence-permitted position around the corresponding N-containing ring, provided that in instances 2) where there is a double bond between the nitrogen atom of either N-containing ring and an adjacent carbon atom thereof, R4 is nothing, provided, if there is no such nitrogen double bond, the corresponding R4 is selected from the group consisting of H, OH, and C1-C12 alkyl groups; or 3) either or both of the N-containing rings are free of non-fused double bonds and each R4 is independently selected from the group consisting of H, OH, and C1-C12 alkyl groups.
 19. The composition of claim 18, said structure being


20. The composition of claim 1, said therapeutic compound having the structure


21. The composition of claim 1, said therapeutic compound having the structure

where the —O—R3 groups may be independently located at any position on the terminal phenyl groups, where each R1 is independently selected from the group consisting of nothing, H, OH, and C1-C12 alkyl groups, each R2 is independently selected form the group consisting of H, OH, and C1-C12 alkyl groups, each R3 group is independently selected from the group consisting of C1-C12 alkyl groups, and substituted or unsubstituted phenyl groups, and wherein the designation

refers to the fact that there may optionally be: 1) one or two non-fused double bonds at one or two valence-permitted positions around either or both of the six-membered, N-containing rings; 2) a double bond between either or both of the N-containing rings and the adjacent carbons of the central moiety, with or without an additional non-fused double bond at any valence-permitted position around the corresponding N-containing ring; or 3) either or both of the N-containing rings are free of non-fused double bonds and each R1 is independently selected from the group consisting of H, OH, and C1-C12 alkyl groups.
 22. The composition of claim 21, wherein said structure is


23. The composition of claim 1, said therapeutic compound having the structure

where the —O—R3 groups may be independently located at any position on the terminal phenyl groups, where each R1 is independently selected from the group consisting of nothing, H, OH, and C1-C12 alkyl groups, each R2 is independently selected form the group consisting of H, OH, and C1-C12 alkyl groups, each R3 group is independently selected from the group consisting of C1-C12 alkyl groups, and substituted or unsubstituted phenyl groups, and the phenoxy group may be substituted at any position on the benzyl ring, and wherein the designation

refers to the fact that there may optionally be: 1) one or two non-fused double bonds at one or two valence-permitted positions around either or both of the six-membered, N-containing rings; 2) a double bond between either or both of the N-containing rings and the adjacent carbons of the central moiety, with or without an additional non-fused double bond at any valence-permitted position around the corresponding N-containing ring; or 3) either or both of the N-containing rings are free of non-fused double bonds and each R1 is independently selected from the group consisting of H, OH, and C1-C12 alkyl groups.
 24. The composition of claim 23, wherein said structure is


25. The composition of claim 1, said therapeutic compound having the structure

wherein each of X1, X2, and X3 is independently selected from the group consisting of nothing, H, OH, C1-C12 alkyl, alkenyl, and alkynyl groups, C1-C12 alkoxy and alkoxyphenyl groups, aryl and aryloxy groups, aldehyde and carbaldehyde groups, amines, nitro groups, nitrile groups, C2-C6 carboxylic acid groups, boronic groups, sulfur groups, and amino acids, where any of the aforementioned may be substituted with N, S, O, B, or halogen atoms, Z is the carbon atom of said methine group, X9 is OH, C1-C12 alkyl, alkenyl, and alkynyl groups, C1-C12 alkoxy and alkoxyphenyl groups, aryl and aryloxy groups, aldehyde and carbaldehyde groups, amines, nitro groups, nitrile groups, C2-C6 carboxylic acid groups, boronic groups, sulfur groups, and amino acids, where any of the aforementioned may be substituted with N, S, O, B, or halogen atoms, each X3 is attached at any position around the corresponding terminal phenyl moieties of the β-carboline groups, each Y is independently nothing, C1-C12 alkyl, alkenyl, and alkynyl groups, C1-C12 alkoxy and alkoxyphenyl groups, aryl and aryloxy groups, aldehyde groups, amines, nitro groups, nitrile groups, C2-C6 carboxylic acid groups, boronic groups, sulfur groups, and amino acids, where any of the aforementioned may be substituted with N, S, O, B, or halogen atoms, Y is a C1-C12 group composed of C, CH, and/or CH2 atoms or groups, M is selected from the group consisting of Structure IIIA, nothing, OH, C1-C12 alkyl, alkenyl, and alkynyl groups, C1-C12 alkoxy and alkoxyphenyl groups, aryl and aryloxy groups, aldehyde groups, amines, nitro groups, nitrile groups, C2-C6 carboxylic acid groups, boronic groups, sulfur groups, and amino acids, where any of the aforementioned may be substituted with N, S, O, B, or halogen atoms, each of X4, X5, X6, X7, and X8 of Structure IIIA is attached at any position around the A ring and is independently selected from the group consisting of nothing, H, OH, C1-C12 alkyl, alkenyl, and alkynyl groups, C1-C12 alkoxy and alkoxyphenyl groups, aryl and aryloxy groups, aldehyde groups, amines, nitro groups, nitrile groups, C2-C6 carboxylic acid groups, boronic groups, sulfur groups, and amino acids, where any of the aforementioned may be substituted with N, S, O, B, or halogen atoms, the designation

in the A ring refers to the fact that there may optionally be 0, 1, 2, or 3 double bonds, and wherein the designation

in connection with the two B rings refers to the fact that there may optionally be: 1) one or two non-fused double bonds at one or two valence-permitted positions around either or both of the B rings, 2) a double bond between either or both of the B rings and Y or Z, with or without an additional non-fused double bond at any valence-permitted position around the corresponding N-containing ring.
 26. The composition of claim 25, wherein M is the 1A ring, both of X1 are nothing, both of X3 are methoxy, 2 of X4, X5, X6, X7, and X8 are H, at least one of X4, X5, X6, X7, and X8 is selected from the group consisting of H, —OH, methoxy, ethoxy, phenoxy, C2-C5 alkenyl groups, F, and C1, with the provisos that: 1) when one or more of X4, X5, X6, X7, and X8 is/are F or C1, the remainder of X4, X5, X6, X7, and X8 are all H; 2) only one of X4, X5, X6, X7, and X8 may be phenoxy, and in such case, the remainder of X4, X5, X6, X7, and X8 are all H.
 27. The composition of claim 25, said therapeutic compound selected from the group consisting of


28. An anti-cancer composition comprising a therapeutic compound which is a reaction product of an aldehyde compound and harmaline, said reaction product comprising two harmaline moieties covalently bonded to a single linker moiety derived from said aldehyde compound, said composition including at least one other ingredient with said reaction product.
 29. The composition of claim 28, said aldehyde compound selected from the group consisting of vanillin, cinnamaldehyde, orthovanillin, phenoxy benzaldehyde, and mixtures thereof.
 30. The composition of claim 28, said therapeutic compound having the general structure II:

where X10 is —CH═CH—, X11, X12, X13, and X14 are each independently selected from the group consisting of H, —OH, methoxy, ethoxy, and phenoxy, F, and C1, with the provisos that: 1) at least one of X12, X13, or X14 is H; 2) when one or more of X11, X12, X13, or X14 is/are F or C1, the remainder of the X11, X12, X13, and X14 are all H; 3) a phenoxy group is present only at X12, and X11, X13, and X14 are all H, and 4) if a methoxy or ethoxy is present, at least one such methoxy or ethoxy must be at either the 2 or 3 position, wherein the designation

in connection with the two N-containing rings refers to the fact that there may optionally be: 1) one or two non-fused double bonds at one or two valence-permitted positions around either or both of the N-containing rings; 2) a double bond between either or both of the N-containing rings and the adjacent carbon atoms, with or without an additional non-fused double bond at any valence-permitted position around the corresponding N-containing ring; or 3) either or both of the N-containing rings are free of non-fused double bonds, and each X1 is independently selected from the group consisting of H, OH, and C1-C12 alkyl groups.
 31. A compound having a pair of fused tricyclic moieties bound together by a single linker moiety, where one of the rings of each of the fused tricyclic moieties is a N-containing ring, said linker moiety providing bonding branches from a single non-metal atom forming at least a part of the linker moiety, such that said fused tricyclic moieties are bonded to said linker through said single atom, said linker including a methine group, said single non-metal atom being the carbon atom of said methine group, with the proviso that said compound does not have two harmaline moieties or two harmine moieties, with a linker moiety of benzaldehyde or p-nitro benzaldehyde.
 32. The compound of claim 31, said linker moiety derived from an aldehyde compound.
 33. The compound of claim 32, said linker moiety derived from a compound having the structure

where the substituents may be located at any position around the ring, R1′ is a C1-C12 aldehyde, R2′-R5′ are independently and selectively taken from the group consisting of H, OH, C1-C12 alkyl groups, C2-C12 alkenyl groups, C1-C12 alkoxy groups, C1-C12 aldehyde groups, acetate, isobutyrate, phenyl, phenoxy, benzyloxy, C2-C6 alkyl esters, halo, and nitro, where the dotted bond lines in the six-membered ring represent that the six-membered ring may be cyclohexane, or have one, two, or three carbon-carbon double bonds, or a C1-C12 alkyl or C2-C12 alkenyl aldehyde, said single atom being the carbonyl carbon of said aldehyde group.
 34. The compound of claim 31, said tricyclic moieties each being β-carboline moieties, where each of the β-carboline moieties is independently selected and derived from compounds having the structure

where the numbered 6-member fused ring is a N-heterocycle with a single N atom at any of the positions 2-5, and the R6 substituents may be located at any ring position, R5′ is H or C1-C12 alkoxy, and R6′ is H, a C1-C12 alkyl, or a C1-C12 carboxylic acid.
 35. The compound of claim 31, said linker being an aldehyde moiety selected from the group consisting of moieties of vanillin, benzaldehyde, cinnamaldehyde, cuminaldehyde, orthovanillin, vanillin isobutyrate, phenoxy benzaldehyde, and mixtures thereof.
 36. The compound of claim 31, said compound having two harmaline moieties and a single linker moiety derived from an aldehyde compound.
 37. The compound of claim 36, said aldehyde moiety selected from the group consisting of moieties of vanillin, benzaldehyde, cinnamaldehyde, cuminaldehyde, orthovanillin, vanillin isobutyrate, phenoxy benzaldehyde, and mixtures thereof.
 38. The compound of claim 31, said compound having two harmaline moieties and a cinnamaldehyde moiety, and having a molecular weight of approximately
 542. 39. The compound of claim 31, said compound having the structure

and the dimers, isomers, and tautomers thereof, where the —O—R3 groups may be independently located at any position on the terminal phenyl groups, where each R1 is independently selected from the group consisting of nothing, H, OH, and C1-C12 alkyl groups, each R2 is independently selected form the group consisting of H, OH, and C1-C12 alkyl groups, each R3 group is independently selected from the group consisting of C1-C12 alkyl groups, and substituted or unsubstituted phenyl groups, and wherein the designation

refers to the fact that there may optionally be: 1) one or two non-fused double bonds at one or two valence-permitted positions around either or both of the six-membered, N-containing rings; 2) a double bond between either or both of the N-containing rings and the adjacent carbons of the central moiety, with or without an additional non-fused double bond at any valence-permitted position around the corresponding N-containing ring; or 3) either or both of the N-containing rings are free of non-fused double bonds and each R1 is independently selected from the group consisting of H, OH, and C1-C12 alkyl groups.
 40. The compound of claim 39, said structure being


41. The compound of claim 31, said compound comprising two harmaline moieties and one vanillin moiety.
 42. The compound of claim 31, said compound having the structure

where each R4 is independently selected from the group consisting of nothing, H, OH, and C1-C12 alkyl groups, each R5 is independently selected from the group consisting of H, OH, and C1-C12 alkyl groups, each R6 group is independently located at any position around the corresponding terminal phenyl group, or at either of the two open positions of the two N-containing rings, and is selected from the group consisting of C1-C12 alkoxy groups, H, OH, and substituted or unsubstituted phenyl groups, R7 and R8 are attached at any position around the benzene ring and are independently selected from the group consisting of H, OH, and C1-C12 alkoxy groups, with the proviso that R7 and R8 are not both H, R7 is OH, R8 is a C1-C12 alkoxy group, and each R9 is independently selected from the group consisting of H, OH, and C1-C12 alkyl groups, and wherein the designation

refers to the fact that there may optionally be: 1) zero, one, or two non-fused double bonds at one or two valence-permitted positions around either or both of the six-membered, N-containing rings; 2) a double bond between either or both of the N-containing rings and the adjacent carbons of the central moiety, with or without an additional non-fused double bond at any valence-permitted position around the corresponding N-containing ring, provided that in instances 2) where there is a double bond between the nitrogen atom of either N-containing ring and an adjacent carbon atom thereof, R4 is nothing, provided, if there is no such nitrogen double bond, the corresponding R4 is selected from the group consisting of H, OH, and C1-C12 alkyl groups; or 3) either or both of the N-containing rings are free of non-fused double bonds and each R4 is independently selected from the group consisting of H, OH, and C1-C12 alkyl groups.
 43. The compound of claim 42, said structure being


44. The compound of claim 31, said compound having the structure


45. The compound of claim 31, said compound having the structure

where the —O—R3 groups may be independently located at any position on the terminal phenyl groups, where each R1 is independently selected from the group consisting of nothing, H, OH, and C1-C12 alkyl groups, each R2 is independently selected form the group consisting of H, OH, and C1-C12 alkyl groups, each R3 group is independently selected from the group consisting of C1-C12 alkyl groups, and substituted or unsubstituted phenyl groups, and wherein the designation

refers to the fact that there may optionally be: 1) one or two non-fused double bonds at one or two valence-permitted positions around either or both of the six-membered, N-containing rings; 2) a double bond between either or both of the N-containing rings and the adjacent carbons of the central moiety, with or without an additional non-fused double bond at any valence-permitted position around the corresponding N-containing ring; or 3) either or both of the N-containing rings are free of non-fused double bonds and each R1 is independently selected from the group consisting of H, OH, and C1-C12 alkyl groups.
 46. The compound of claim 45, wherein said structure is


47. The compound of claim 31, said compound having the structure

where the —O—R3 groups may be independently located at any position on the terminal phenyl groups, where each R1 is independently selected from the group consisting of nothing, H, OH, and C1-C12 alkyl groups, each R2 is independently selected form the group consisting of H, OH, and C1-C12 alkyl groups, each R3 group is independently selected from the group consisting of C1-C12 alkyl groups, and substituted or unsubstituted phenyl groups, and the phenoxy group may be substituted at any position on the benzyl ring, and wherein the designation

refers to the fact that there may optionally be: 1) one or two non-fused double bonds at one or two valence-permitted positions around either or both of the six-membered, N-containing rings; 2) a double bond between either or both of the N-containing rings and the adjacent carbons of the central moiety, with or without an additional non-fused double bond at any valence-permitted position around the corresponding N-containing ring; or 3) either or both of the N-containing rings are free of non-fused double bonds and each R1 is independently selected from the group consisting of H, OH, and C1-C12 alkyl groups.
 48. The compound of claim 47, wherein said structure is


49. The compound of claim 31, said compound selected from the group consisting of


50. A method of treating a human cancer patient comprising the step of administering to the patient an effective amount of a composition in accordance with claim
 1. 51. The method of claim 50, said compound comprising two harmaline moieties covalently bonded to a single moiety derived from an aldehyde compound.
 52. The method of claim 51, said aldehyde selected from the group consisting of vanillin, benzaldehyde, cinnamaldehyde, orthovanillin, phenoxy benzaldehyde, and mixtures thereof. 